Prophylactic Use of Theta Defensins

Abstract

Compositions and methods are described that provide safe and effective prophylactic treatment of a wide range of microbial pathogens. Such compositions and methods incorporate theta defensins and theta defensin analogs, which have been found to have broad-spectrum antibacterial effects, maintain effective concentrations in the bloodstream for extended periods of time, and are safe to administer at high doses.

Description

[Technical field]

[0001] The technical field of this invention is the prophylactic treatment of microbial infections. [Background technology]

[0002] The Background Section contains information that may be useful in understanding the present invention. No admission is made that the information provided in the Background Section is prior art or relevant art to the presently claimed invention, nor is any publication specifically or implicitly admitted to be prior art.

[0003] Prophylactic treatment for the prevention of infectious diseases is a fundamental part of public health policy. Such treatment is provided in the absence of ongoing disease. Thus, considerations in prevention include that the risk of side effects from the prophylactic treatment outweigh the risk of contracting and being adversely affected by the disease that is being prevented. Thus, for a prophylactic treatment to be useful, it should be effective while at the same time providing a margin of safety with an effective dose that exceeds the effective dose of the treatment used to address ongoing symptomatic disease.

[0004] Vaccines are a well-known example of preventive therapy. Vaccination with an appropriate vaccine induces an antibody response against a specific pathogen in the vaccinated individual. Current vaccine formulations are highly effective and pose very low risks. For example, currently, over 400 million doses of vaccine targeting the causative agent of COVID-19 (SARS-CoV-2) have been administered in the United States, and the vaccines are highly effective in reducing infection (about 94% for mRNA-based formulations) and appear to prevent severe disease in rare breakthrough infections. However, close monitoring by the Vaccine Adverse Event Reporting System reports that all-cause mortality after vaccination is only about 0.0021%.

[0005] However, such preventive vaccines are generally highly specific. As a result, there is an inevitable lag between the recognition of a new pathogen and the development, testing, production, and distribution of a safe and effective vaccine targeting it. Although newer approaches such as the use of mRNA vaccines have shortened this lag period, it is still considerable. Furthermore, individuals with poor immune responses are less likely to develop an antibody response sufficient to prevent or reduce the severity of infection after vaccination. Such at-risk individuals may require additional vaccinations or may not develop a protective antibody response at all.

[0006] Thus, there remains a need for safe prophylactic compositions and methods that provide effective protection against a broad range of pathogens and that are effective in immunocompromised individuals. Summary of the Invention

[0007] The present subject matter provides compositions and methods using theta-defensins and functional analogs of theta-defensins for prophylactic protection against microbial pathogens, which have broad-spectrum antibacterial activity, persist in the body after administration, and have been found to be safe at high doses.

[0008] An embodiment of the inventive concept includes a method of providing prophylactic treatment against a microbial infection by identifying an individual in need of prophylactic treatment against a microbial infection and administering a θ-defensin (e.g., RTD-1 (SEQ ID NO: 1)) or a θ-defensin analog to the individual using a protocol that maintains at least 50% of the peak plasma concentration of the θ-defensin or θ-defensin analog for at least 4 hours after administration. First, an effective θ-defensin or θ-defensin analog is identified that maintains at least 50% of the peak plasma concentration for at least 4 hours after administration. After identifying an individual in need of prophylactic treatment, an effective θ-defensin or θ-defensin analog is administered to the individual in an amount sufficient such that at least 50% of the peak plasma concentration is sufficient to exert an antimicrobial effect against the microorganism against which prevention is desired. In some embodiments, such individuals are identified as high-risk individuals, such as newborns, premature infants, children with underdeveloped or partially developed immune systems, elderly individuals, individuals recovering from surgery, individuals recovering from accidental injury, individuals with cancer, immunocompromised individuals, individuals who have received an organ, bone marrow, or stem cell transplant, individuals undergoing or recovering from chemotherapy, individuals undergoing or recovering from radiation therapy, individuals undergoing or recovering from immunotherapy, individuals undergoing or recovering from immunosuppressive therapy, individuals with underlying heart disease and / or cardiac damage, individuals with underlying renal disease and / or renal damage, individuals with underlying liver disease and / or liver damage, individuals with underlying lung disease and / or lung damage, individuals with obesity, and / or identified based on exposure to others within a distance where the microorganism may be transmitted. The microorganism of interest in such methods may be a virus (e.g., SARS-CoV-2), a bacterium, a fungus, or a protozoan. In such methods, the θ-defensin or θ-defensin analog is administered at up to 15 mg / kg. The θ-defensin or θ-defensin analog can be administered as a single dose, such as a dose sufficient to provide between 0.1 mg / kg and 15 mg / kg of theta-defensin or θ-defensin analog to an individual in need of treatment.Alternatively, the θ-defensin or θ-defensin analog can be administered in two or more doses (e.g., at a frequency of from once every 12 hours to once every 48 hours), where each individual dose is sufficient to provide from 0.1 mg / kg to 15 mg / kg of the θ-defensin or θ-defensin analog to the individual in need of treatment.

[0009] Embodiments include formulations for providing prophylactic treatment of microbial infections comprising one or more effective θ-defensins and / or effective θ-defensin analogs, as well as the use of one or more effective θ-defensins and / or effective θ-defensin analogs in the preparation of such formulations.

[0010] Various objects, features, aspects and advantages of the present subject matter will become more apparent from the following detailed description of preferred embodiments, taken in conjunction with the accompanying drawings in which like numerals represent like elements and in which: [Brief description of the drawings]

[0011] [Figure 1A] 1A-1D show typical results of plasma concentration versus time studies in different species. Typical mean (SD) plasma concentration-time profiles of RTD-1 (SEQ ID NO: 1) in mice (n=4 / time point) are shown. [Figure 1B] 1A-1D show typical results of plasma concentration versus time studies in different species. Typical mean (SD) plasma concentration-time profiles of RTD-1 (SEQ ID NO: 1) in rats (n=6 / time point) are shown. [Figure 1C] 1A-1D show typical results of plasma concentration versus time studies in different species. Typical mean (SD) plasma concentration-time profiles of RTD-1 (SEQ ID NO: 1) in cynomolgus monkeys (n=2-12 / treatment group) are shown. [Figure 1D]1A-1D show typical results of plasma concentration versus time studies in different species. Typical mean (SD) plasma concentration-time profile of RTD-1 (SEQ ID NO: 1) in vervet monkeys (n=1) after IV administration of a single dose is shown. [Figure 2A] Representative mean (SD) plasma concentration versus time profiles in rats after repeated daily iv dosing of 5 mg / kg / day for 7 days are shown. [Figure 2B] Representative mean (SD) plasma concentration versus time profiles in rats after repeated daily iv dosing of 10 mg / kg / day for 7 days are shown. [Figure 3A] FIG. 1 shows typical mean (SD) plasma concentration versus time profiles in cynomolgus monkeys after repeated iv administration of RTD-1 (SEQ ID NO: 1) at 5 mg / kg / day in the recovery group (n=4). [Figure 3B] FIG. 1 shows typical mean (SD) plasma concentration versus time profiles in cynomolgus monkeys after repeated iv administration of RTD-1 (SEQ ID NO: 1) at 10 mg / kg / day in the recovery group (n=4). [Figure 3C] FIG. 1 shows typical mean (SD) plasma concentration versus time profiles in cynomolgus monkeys after repeated iv administration of RTD-1 (SEQ ID NO: 1) at 15 mg / kg / day in the recovery group (n=4). [Figure 3D] FIG. 1 shows typical mean (SD) plasma concentration versus time profiles in cynomolgus monkeys after repeated iv administration of RTD-1 (SEQ ID NO: 1) at 15 mg / kg in the recovery group (n=4). [Figure 4A] 1 shows typical results of an assessment of dose proportionality of Cmax in cynomolgus monkeys after administration of a single dose of RTD-1 (SEQ ID NO: 1) (0.3, 1, 3, 10, or 15 mg / kg). [Figure 4B] 1 shows typical results of an assessment of dose proportionality of AUC0-∞ in cynomolgus monkeys after administration of a single dose of RTD-1 (SEQ ID NO: 1) (0.3, 1, 3, 10, or 15 mg / kg). [Figure 5A]Figure 1 shows typical results of an interspecies allometric correlation study. The plot was generated based on the results of the RTD-1 (SEQ ID NO: 1) single dose PK study. The dashed line represents the predicted CL (6.44 L / h) for a 70 kg adult. [Figure 5B] Figure 1 shows typical results of an interspecies allometric correlation study. The plot was generated based on the results of a single dose PK study of RTD-1 (SEQ ID NO: 1). The dashed line represents the predicted value of Vss (28.0 L) for a 70 kg adult. [Figure 6] Typical mean (SD) density and distribution of 14C-RTD-1 (SEQ ID NO: 1) in tissues and organs 1 and 24 hours after intravenous administration to female rats. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present subject matter provides compositions and methods that provide safe and effective prophylactic treatment of a wide range of microbial pathogens. The inventors have discovered that theta defensins can maintain effective concentrations in the bloodstream for extended periods of time and are safe to administer in high doses.

[0013] It should be appreciated that the disclosed technology provides many advantageous technical effects, including providing safe and effective prevention of microbial infections in high-risk populations without the need for the development of biospecific formulations.

[0014] The following description contains information that may be useful in understanding the present invention. No admission is made that the information provided herein is prior art or relevant art to the presently claimed invention, nor is any publication specifically or implicitly admitted to be prior art.

[0015] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. In the event that a definition or use of a term in an incorporated reference is inconsistent with or contrary to the definition of that term provided herein, the definition of that term provided herein shall apply and the definition of that term in the reference shall not apply.

[0016] 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 should be understood to be modified in some cases by the term "about". Thus, in some embodiments, the numerical parameters set forth in the written description and accompanying 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 number of 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 ​​presented in some embodiments of the present invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0017] 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 in this description, the meaning of "in" includes "in" and "on," unless the context clearly dictates otherwise.

[0018] The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of individually referring to each separate value falling within the range. Unless otherwise indicated herein, each separate 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. Any and all examples provided with respect to specific embodiments herein, or the use of exemplary language (e.g., "such as"), are intended merely to better clarify the invention and do not limit 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.

[0019] Groupings of alternative elements or embodiments of the invention disclosed herein should not 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 such inclusion or deletion occurs, the specification is deemed herein to include the modified group, thereby satisfying the written description of all Markush groups used in the appended claims.

[0020] The following description provides a number of exemplary embodiments of the inventive subject matter. Although each embodiment represents a single combination of the inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus, if one embodiment includes elements A, B, and C, and a second embodiment includes elements B and D, the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.

[0021] The inventors have found that theta defensins (e.g., RTD-1 (SEQ ID NO: 1)) are very well tolerated and exhibit good pharmacokinetics with rapid distribution and retention of effective concentrations for extended periods (e.g., 2, 3, 4, 5, 6, 8, 12, 23, 36, 48 hours or more) following administration, indicating a long-term effect useful in prophylactic treatment. Similarly, studies have shown that administration of large amounts (e.g., up to or greater than 15 mg / kg) of theta defensins is well tolerated and has a safety profile suitable for prophylactic use, particularly in vulnerable and high-risk individuals.

[0022] Given the activity of theta defensins against a wide range of microbial (e.g., viral, bacterial, fungal, protozoan) infections, the inventors believe that such theta defensins may be useful in the prophylactic treatment of such microbial infections. Given the observed high level of tolerability, the inventors believe that such prophylactic treatments would be particularly useful in high-risk populations that are particularly susceptible to such infections. Such high-risk populations include, but are not limited to, neonates, premature infants, children with underdeveloped or partially developed immune systems, elderly individuals, individuals recovering from surgery, individuals recovering from accidental injury, individuals with cancer, immunocompromised individuals, individuals who have received organ, bone marrow, or stem cell transplants, individuals undergoing or recovering from chemotherapy, individuals undergoing or recovering from radiation therapy, individuals undergoing or recovering from immunotherapy, individuals with underlying heart disease and / or cardiac injury, individuals with underlying renal disease and / or kidney injury, individuals with underlying liver disease and / or liver injury, individuals with underlying lung disease and / or lung injury, and obese individuals.

[0023] In other embodiments, a high-risk individual may be someone who is at particular risk of exposure to a microbial pathogen, for example, by being within a certain distance from a source of infection (e.g., a symptomatic or asymptomatic infected person) through which the microorganism may be transmitted. This distance may vary depending on the microorganism and its mode of transmission. For example, such distance may range from direct or close contact from the source of the pathogen to about 2 meters or more (in the case of aerosol transmission). Such high-risk individuals may be, for example, individuals whose occupation requires contact with or close proximity to potentially infected individuals (e.g., health care providers, teachers, emergency responders, law enforcement, etc.). Alternatively, high-risk individuals may be individuals who anticipate social interactions that may result in contact with or exposure to infected individuals.

[0024] Although the theta defensin RTD-1 (SEQ ID NO: 1) is referred to herein, the inventors contemplate that other theta defensins may be effective in the methods of the inventive concepts. Within the context of the present application, an effective θ-defensin is one that exhibits bactericidal and / or microbistatic (i.e., halts microbial proliferation) effects at concentrations achievable in humans following administration by oral administration, injection, infusion, inhalation, and / or application to the eye or mucosa. Such effects may be demonstrated against viral, bacterial, fungal, and / or protozoan pathogens. Similarly, the methods of the inventive concepts may utilize an effective amount of an effective θ-defensin. Within the context of the present application, an effective amount is an amount of an effective θ-defensin sufficient to maintain a bactericidal and / or microbistatic effect in an individual administered an effective amount of the effective θ-defensin for a period of 4 hours, 8 hours, 12 hours, 24 hours, 48 ​​hours, 3 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, or more than 1 month after administration. In some embodiments, an effective amount of an effective theta-defensin or effective theta-defensin analog can be 10%, 20%, 30%, 40%, 50%, or more of the peak serum concentration following administration.

[0025] Examples of suitable θ-defensins include RTD-1 (SEQ ID NO:1), RTD-2 (SEQ ID NO:2), RTD-3 (SEQ ID NO:3), RTD-4 (SEQ ID NO:4), RTD-5 (SEQ ID NO:5), RTD-6 (SEQ ID NO:6), BTD-1 (SEQ ID NO:7), BTD-2 (SEQ ID NO:8), BTD-3 (SEQ ID NO:9), BTD-4 (SEQ ID NO:10), BTD-5 (SEQ ID NO:11), BTD-6 (SEQ ID NO:12), BTD-7 (SEQ ID NO:13), BTD-8 (SEQ ID NO:14), BTD-9 (SEQ ID NO:15), and / or BTD-10 (SEQ ID NO:16). Also contemplated is a peptide derived from alternative splicing of the human protodefensin HTDp, which is naturally transcribed but not translated (SEQ ID NO:17). In some embodiments, more than one θ-defensin can be used in the method of the present inventive concept.

[0026] Instead of or in addition to naturally occurring θ-defensins, embodiments of the inventive concept may use one or more effective θ-defensin analogs. The term θ-defensin analog refers to a cyclic peptide having about 40%, 50%, 60%, 70%, 80%, 90% or more sequence identity with a natural θ-defensin peptide sequence. A θ-defensin analog may incorporate one, two, three or more core features of a natural θ-defensin. Exemplary core features include a cyclic structure, the presence of one, two, three or more disulfide bonds within the peptide (e.g., between pairs of cysteines in the analog), a positive charge when in solution under physiological conditions, and the presence of a β-pleated sheet secondary structure. Such θ-defensin analogs can include 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 amino acids, and in some embodiments, can incorporate non-naturally occurring amino acids. Analogs of θ-defensins can include one or more L-amino acids, one or more D-amino acids, and / or mixtures of L- and D-amino acids. In some embodiments, non-peptide bonds can be utilized between adjacent amino acid residues of the θ-defensin analogs. θ-defensin analogs can exhibit one or more deletions or substitutions of amino acids of a native θ-defensin sequence. Such substitutions can be conservative (e.g., the substituted amino acids retain the charge, hydrophobicity, hydrophilicity, and / or steric properties of the native amino acids). In some embodiments, theta-defensin analogs can include grafting or conjugation of non-peptide moieties (e.g., polyethylene glycol and / or other hydrophilic polymers, cell receptor targeting moieties, and / or moieties that aid in processing / purification).

[0027] Within the context of this application, an effective θ-defensin analog is one that exhibits bactericidal and / or microbistatic (i.e., stops microbial proliferation) effects at concentrations achievable in humans following administration by oral administration, injection, infusion, inhalation, and / or application to the eye or mucosa. Such effects may be demonstrated against viral, bacterial, fungal, and / or protozoan pathogens. Similarly, the methods of the inventive concept may utilize an effective amount of an effective θ-defensin analog. Within the context of this application, an effective amount is an amount of an effective θ-defensin analog sufficient to maintain a bactericidal and / or microbistatic effect in an individual administered an effective amount of the effective θ-defensin for a period of 12 hours, 24 hours, 48 ​​hours, 3 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, or more than 1 month after administration.

[0028] Examples of suitable θ-defensin analogs include peptide 1 (SEQ ID NO:18), peptide 2 (SEQ ID NO:19), peptide 3 (SEQ ID NO:20), peptide 4 (SEQ ID NO:21), peptide 5 (SEQ ID NO:22), peptide 6 (SEQ ID NO:23), peptide 7 (SEQ ID NO:24), peptide 8 (SEQ ID NO:25), peptide 9 (SEQ ID NO:26), peptide 10 (SEQ ID NO:27), peptide 11 (SEQ ID NO:28), peptide 12 (SEQ ID NO:29), peptide 13 (SEQ ID NO:30), peptide 14 (SEQ ID NO:31), and peptide 15 (SEQ ID NO:32). In some embodiments, more than one effective θ-defensin analog can be used in the methods of the present inventive concept. In some embodiments, a combination of one or more effective θ-defensins and one or more effective θ-defensin analogs can be used.

[0029] It should be understood that the θ-defensins are very similar in size, secondary and tertiary structure, charge, degree of hydrophobicity, and resistance to proteolysis. Therefore, it is reasonable to expect that the θ-defensins will follow similar degradation and excretion pathways after administration. Similarly, the θ-defensin analogs described herein are essentially similar in structure, charge, and degree of hydrophobicity to naturally occurring θ-defensins, and it is reasonable to expect that they will utilize similar degradation and excretion pathways. Therefore, suitable θ-defensins and / or θ-defensin analogs that are effective in providing pharmacokinetic properties equivalent to or better than RTD-1 (SEQ ID NO: 1) can be easily identified, for example, through and / or by reference to animal tests as described below. Similarly, suitable θ-defensins and / or θ-defensin analogs that exert antibacterial effects equivalent to or better than RTD-1 (SEQ ID NO: 1) can be easily identified, for example, through and / or by reference to animal models of microbial disease. In some embodiments, effective θ-defensins and / or θ-defensin analogs can have comparable or improved pharmacokinetic properties and safety profiles compared to RTD-1 (SEQ ID NO: 1), and therefore can be used for the prophylactic treatment of microbial infections.Similarly, although intravenous administration is described herein, other administration routes may also be suitable.Suitable alternative routes for administration include subcutaneous injection, intraperitoneal injection, injection into cerebrospinal fluid, topical administration, inhalation, and / or oral administration.

[0030] In a typical method of prophylactic treatment, a θ-defensin and / or an effective θ-defensin analog is identified that is effective to provide antimicrobial activity with a plasma concentration sustained for at least 4 hours (i.e., an effective θ-defensin and / or an effective θ-defensin analog). This can be accomplished by experiment (e.g., using an animal model of microbial infection and / or pharmacokinetic studies) or can be determined from historical data. Similarly, an individual in need of prophylactic therapy is identified and an effective θ-defensin and / or an effective θ-defensin analog is administered. Such an effective dose can provide a plasma concentration of an effective θ-defensin and / or an effective θ-defensin analog that is sufficient to exert a bactericidal and / or microbistatic effect for at least 4 hours, 8 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or more. Such methods can provide, for example, an effective θ-defensin and / or effective θ-defensin analog that maintains at least 50% of the peak plasma concentration for at least 4 hours after administration to a person identified as in need of prophylactic treatment to prevent microbial infection, where the 50% peak plasma concentration of the effective θ-defensin and / or effective θ-defensin analog is a dose sufficient to exert an antimicrobial effect against a target microorganism. Suitable routes of administration include injection (e.g., subcutaneous, intramuscular, or peritoneal injection), inhalation, and / or topical (e.g., to a mucosa).

[0031] Compounds useful for preventive therapy should be considered very safe to use. The safety and pharmacokinetic parameters of RTD-1 (SEQ ID NO: 1), which may be considered a prototypic θ-defensin, are shown below. Single and repeated dose studies conducted in rats and cynomolgus monkeys demonstrated an excellent safety profile with intravenous administration of RTD-1 (SEQ ID NO: 1). Repeated administration of RTD-1 (SEQ ID NO: 1) was well tolerated in rats at doses up to 10 mg / kg, and therefore the NOAEL in rats was set at 10 mg / kg / day. Treatment-related mortality and adverse clinical signs, including cold to the touch, abnormal color, lameness, severe dehydration, and tremors, were observed in rats treated with 20 mg / kg. Single and repeated doses once daily of RTD-1 (SEQ ID NO: 1) were tolerated in cynomolgus monkeys up to 15 mg / kg / day without major treatment-related adverse findings or toxicity. Considering the lack of adverse findings, the NOAEL was set at 15 mg / kg / day in cynomolgus monkeys. The NOAEL in cynomolgus monkeys was set at a higher dose compared to rats, demonstrating that RTD-1 (SEQ ID NO: 1) is better tolerated in cynomolgus monkeys. Most of the changes observed in hematological, serum chemical, and coagulation parameters in both rats and cynomolgus monkeys were determined to be non-adverse due to their small magnitude, lack of consistency between the two species, lack of dose dependency, and / or reversibility by the end of the recovery period. Thus, these data demonstrate the safety of intravenous administration of RTD-1 (SEQ ID NO: 1) at doses up to 10 mg / kg / day in rats and up to 15 mg / kg / day in cynomolgus monkeys.

[0032] The intravenous PK of RTD-1 (SEQ ID NO: 1) after single and repeated ascending dose administration in multiple species is characterized by extensive tissue distribution and prolonged elimination. The volume of distribution (Vss) normalized to body weight in animals administered 5 mg / kg RTD-1 (SEQ ID NO: 1) varied by species, being 1,048, 1,461, and 550 mL / kg in mice, rats, and cynomolgus monkeys, respectively. The relatively large Vss indicates that RTD-1 (SEQ ID NO: 1) is distributed extensively in tissues. Biodistribution studies confirmed extensive tissue distribution, especially in the liver. The long elimination half-life (47.2 hours) observed in the recovery group of cynomolgus monkeys suggests tissue redistribution.

[0033] Analysis of single and multiple ascending dose studies in rats and monkeys showed a greater increase in AUC0-∞ and Cmax than the dose ratio, suggesting nonlinear PK. Some therapeutic proteins exhibit nonlinear PK mediated by different mechanisms. For example, exenatide and recombinant human interferon (IFN) (e.g., IFN-β1a) exhibit nonlinear kinetics due to saturation of elimination pathways such as target-mediated drug disposition (TMDD) (24, 25). Alternatively, the nonlinear PK of cyclosporine A and erythropoietin was attributed to tissue binding and receptor saturation in target tissues, respectively (26, 27). In rats in biodistribution studies 14 The broad distribution of C-RTD-1 (SEQ ID NO: 1) could explain the large Vss estimated in preclinical PK studies, suggesting that saturation of the peptide in these tissues could be one of the potential causes of nonlinearity. The dose-dependent decrease in CL observed in rats and cynomolgus monkeys could also explain the nonlinearity, since the greatest accumulation of RTD-1 (SEQ ID NO: 1) occurred in the liver and kidney. In agreement with data from other small peptides (<10 kDa) that are mainly removed by glomerular filtration, a significant amount of 14C-RTD-1 (SEQ ID NO: 1) was recovered in urine (accounting for 7% at 24 h). Furthermore, a relatively significant proportion was recovered in feces (accounting for 4% at 24 h), indicating that elimination of RTD-1 (SEQ ID NO: 1) occurs via renal and biliary excretion (28, 29). Thus, the nonlinear PK of RTD-1 (SEQ ID NO: 1) may be due to saturation of uptake and / or excretion transporters present in the liver or kidney. However, the definitive role of hepatic / renal transporters in the distribution and elimination of RTD-1 (SEQ ID NO: 1) requires further investigation.

[0034] Interspecies allometric scaling provides a method for extrapolating PK data from preclinical species to humans and is commonly used to predict appropriate doses for FIH clinical trials. Because RTD-1 (SEQ ID NO:1) appears to follow linear PK at the HED for efficacy, as evidenced by the dose-proportional increase in AUC0-∞ at low doses (0.3-3 mg / kg) in cynomolgus monkeys, we performed interspecies allometric scaling using simple allometry to predict human PK. For renally excreted macromolecules, human CL can be adequately predicted using simple allometric equations (30). Because three or more preclinical species are typically required to reliably scale parameters to humans, available single-dose data from mice and vervet monkeys were included in the analysis (31). The estimated allometric scaling exponents for CL and Vss were 0.829 and 0.866, respectively, consistent with values ​​reported for other therapeutic proteins, which are 0.65-0.84 for CL and 0.84-1.02 for Vss (32). The target AUC was previously established in a mouse model of LPS-induced ALI, where a single subcutaneous injection of 5 mg / kg or 25 mg / kg RTD-1 (SEQ ID NO:1) resulted in a mean AUC0-∞ of 3,869 ng*h / mL and 9,001 ng*h / mL, respectively, with a significant reduction in airway neutrophil burden and inflammatory cytokines / chemokines without mortality (3). The HED of 0.3 mg / kg daily was determined using the predicted human CL (6.44 L / h) from interspecies allometric scaling and the target AUC for efficacy (from a mouse model of endotoxin-induced ALI). Based on the 10-fold safety factor recommended by the FDA, the predicted first-in-human (FIH) dose in clinical trials is approximately 0.03 mg / kg for adults. (33, 34) This approximation of the dose for FIH studies is predicted to be well below the NOAEL established by preclinical animals and is therefore predicted to be safe in humans.The HED corresponding to the NOAEL in cynomolgus monkeys of 15.9 mg / kg was higher than both the HED corresponding to the NOAEL and LOAEL in rats (3.1 and 11.7 mg / kg, respectively). Because cynomolgus monkeys are physiologically more similar to humans than rats, doses up to 15.9 mg / kg may be tolerated in humans. Furthermore, this indicates that the HED required for efficacy (0.36-0.83 mg / kg) is approximately 19-45 times lower than the HED calculated based on the NOAEL in cynomolgus monkeys, further ensuring safety in humans.

[0035] It should be understood that simple allometry may not account for dose-dependent (non-linear) processes. However, since non-linearity was more evident at higher dose ranges, Applicants believe that at the doses selected for FIH clinical trials, RTD-1 (SEQ ID NO: 1) is predicted to exhibit linear PK. Target AUC Used to Determine FIH Dose 0-∞ It should also be appreciated that was established in a mouse model of LPS-induced ALI and therefore may not reflect the actual target AUC required for efficacy in humans, however, the inventors believe this to be an appropriate animal model for deriving a target AUC for efficacy in the prophylactic treatment of a broad range of microbial infections.

[0036] Pharmacokinetics Mouse study: The mean plasma concentration-time profile after a single dose of RTD-1 (SEQ ID NO: 1) in mice is shown in Figure 1A, and the corresponding pharmacokinetic (PK) parameters calculated by noncompartmental analysis (NCA) are listed in Table 1. Terminal blood draws from each mouse generated pooled PK results. After a single iv bolus dose, RTD-1 (SEQ ID NO: 1) showed a biphasic profile with a relatively short distribution phase followed by a longer elimination phase with a half-life of 6.05 hours.

[0037] [Table 1]

[0038] Rat studies: Mean plasma concentration-time profiles in rats after single (5, 10, or 20 mg / kg) or repeated (5 mg / kg / day or 10 mg / kg / day) administration of RTD-1 (SEQ ID NO: 1) are shown in Figure 1B and Figure 2, respectively, and the corresponding PK parameters are summarized in Table 2. Of a total of 180 infusions from 36 rats, 5 infusions deviated by more than 10% from the intended 20-minute infusion. However, these deviations did not occur on blood PK sampling days and therefore did not affect the PK analysis. Pooled PK results were generated in WinNonlin because data per rat were sparsely sampled. Plasma levels of RTD-1 (SEQ ID NO: 1) were undetectable during the recovery period (day 25) in all rats, except for one female rat that received 10 mg / kg at a concentration of 12.5 ng / mL. Early death of a rat in the 20 mg / kg group precluded PK analysis with repeated dosing at this dose level. Cmax was slightly higher in female compared to male rats, but the difference did not reach statistical significance. Cmax and AUC 0-∞ Although both appear to increase proportionally with dose based on the 95% confidence interval (CI) of the slope that includes 1 (Cmax, Y = 1.110*X + 8.936 [slope 95% CI: 0.7066 to 1.513], R2 = 0.6803; AUC0-∞, Y = 1.543*X + 9.448 [slope 95% CI: 0.4460 to 2.639], R2 = 0.9969), a comprehensive analysis of dose proportionality in rats was limited by the pooled calculation of AUC0-∞ at each dose level and the relatively narrow range of doses tested. AUCτ on day 7 was slightly lower (22% and 19% for the 5 mg / kg and 10 mg / kg groups, respectively) when compared with their AUC0-∞ on day 1, indicating no significant drug accumulation.

[0039] [Table 2]

[0040] Cynomolgus monkey study: Mean plasma concentration-time profiles in cynomolgus monkeys after single or repeated dose administration of RTD-1 (SEQ ID NO: 1) are shown in Figure 1C and Figure 3, respectively. The corresponding PK parameters calculated by NCA are summarized in Table 3. Of a total of 233 infusions from 24 cynomolgus monkeys, 4 infusions deviated from the intended 1-h infusion by more than 10%. However, these deviations did not occur on blood PK sampling days and therefore did not affect the PK analysis. Data from two separate studies involving single and repeated dosing in cynomolgus monkeys were combined in this analysis. Overall, the concentration-time profiles showed a biphasic pattern with a prolonged elimination phase at higher doses. Plasma concentrations of RTD-1 (SEQ ID NO: 1) after a single dose of 0.3 mg / kg or 1 mg / kg were detectable for up to 12 hours after the end of the infusion. In a GLP-compliant 10-day TK study, all animals received 10 iv doses, except for one female monkey at 15 mg / kg, which received a total of 9 doses due to venous access issues. Long-term sampling with cynomolgus monkeys assigned to the recovery group (15 mg / kg) revealed quantifiable plasma levels of RTD-1 (SEQ ID NO: 1) on days 12 (537 ng / mL) and 24 (12.5 ng / mL), indicating that RTD-1 (SEQ ID NO: 1) exhibits a long terminal half-life (Figure 3D). The mean terminal half-life in recovery animals was 47.2 hours compared to 9.53 hours in the main group, which had a shorter sampling period. Cmax and AUC0-∞ were slightly higher in males compared to females, but the differences did not reach statistical significance. Detailed assessment of dose proportionality in cynomolgus monkeys administered single iv doses ranging from 0.3 mg / kg to 15 mg / kg revealed that both Cmax and AUC0-∞ increased more than dose proportionally (Figure 4). 0-∞was dose-proportional at lower doses (0.3 mg / kg to 3 mg / kg) but began to deviate from dose-proportionality at higher doses (5 mg / kg and above) (data not shown). Dose-proportionality assessment at steady state showed that Cmax increased proportionally with dose (Cmax, Y = 0.9553*X + 6.805 [slope 95% CI: 0.5573 to 1.353, R2 = 0.5705]), but AUCτ increased more than proportionally with dose (AUCτ, Y = 1.422*X + 6.745 [slope 95% CI: 1.072 to 1.772, R2 = 0.5705]). 2 = 0.7916]). However, these results were limited by the narrow range of doses tested. Comparison of AUC after single dose (day 1) and multiple dose administration (day 10) revealed statistically significant accumulation at 5 mg / kg and 10 mg / kg, with mean AUCτ being approximately 1.4- and 1.5-fold higher compared to mean AUC0-∞ on day 1 at 5 mg / kg and 10 mg / kg, respectively [5 mg / kg (p = 0.0229) and 10 mg / kg (p = 0.0103)]. There was a trend towards RTD-1 (SEQ ID NO: 1) accumulation at steady state (day 10) at 15 mg / kg, but the difference did not reach statistical significance (p = 0.1879). In the 15 mg / kg group, statistical analysis was not performed with PK parameters calculated on days 4 and 7 due to the small number of animals (n = 2).

[0041] [Table 3]

[0042] Vervet Monkey Study: The mean plasma concentration-time profile of RTD-1 (SEQ ID NO: 1) in vervet monkeys after a single iv bolus administration of RTD-1 (SEQ ID NO: 1) is shown in Figure 1D, and PK parameters are shown in Table 4. After bolus administration, plasma concentrations of RTD-1 (SEQ ID NO: 1) declined monoexponentially. However, plasma concentrations collected after 24 hours were below the lower limit of quantification and therefore excluded from analysis.

[0043] [Table 4]

[0044] Interspecies allometric scaling: Overall, linear regressions of log-transformed CL or Vss against log-transformed body weight (BW) from the four preclinical species yielded reasonable fits, as evidenced by the relatively high r2 (Figure 5). The allometric scaling equations for CL and Vss, respectively, were: Y = 190.1 BW 0.8291 (r2=0.7719) and Y=706.3·BW 0.8663 (r2=0.8853), which resulted in a predicted human CL of 6.44 L / h and a volume of distribution at steady state (Vss) of 28.0 L. Based on target plasma AUC0-∞ of approximately 3,869 ng*h / mL and 9,001 ng*h / mL previously established in a mouse model of endotoxin-induced acute lung injury (ALI), the estimated human equivalent dose (HED) to reach therapeutic efficacy is 24.9 mg to 58.0 mg, or 0.36 mg / kg to 0.83 mg / kg for a 70 kg individual.

[0045] Biodistribution: equivalent to 5mg / kg 14 A biodistribution study was performed to determine the distribution pattern and possible elimination pathways of RTD-1 (SEQ ID NO: 1) in rats following a single iv dose of C-RTD-1 (SEQ ID NO: 1). 14 A widespread distribution of C-RTD-1 (SEQ ID NO: 1) was observed at 1 hour, with the highest density measured in the liver, followed by the kidney (Figure 6). After 1 hour, trace amounts were detected in the urine, skin, leg muscle, eye, and brain. 14 C counts were measured in tissues and organs after 24 hours. 14 The density of C counts was decreased compared to the counts detected at 1 hour, except in urine. 14 The C count increased from traces at 1 hour to 8.5% at 24 hours. 14 Approximately 4% of the C-RTD-1 (SEQ ID NO: 1) dose was collected in the feces at 24 hours, suggesting that the primary route of elimination was via urine, followed by biliary excretion.

[0046] safety Rat Study: The results of the safety evaluation of RTD-1 (SEQ ID NO: 1) administration are provided in detail in the Supplementary Material. In general, single dose administration of up to 10 mg / kg was well tolerated in male and female rats. A total of seven rat deaths were observed during the study. Specifically, we found that one of 18 female rats in the placebo group died on day 6, and further found that one of 16 female rats in the 5 mg / kg dose group died on day 15 of the study. However, the death of the female rat in the 5 mg / kg group was determined to be unrelated to RTD-1 (SEQ ID NO: 1) treatment due to minimal clinical symptoms up to the day of death and the timing of the event. Furthermore, a single iv administration of 20 mg / kg RTD-1 (SEQ ID NO: 1) resulted in treatment-related acute deaths for 5 of 12 rats on day 1. Of the five rats, three male rats were found dead, and one male and one female rat were euthanized due to moribundity.

[0047] After once-daily iv infusion of RTD-1 (SEQ ID NO: 1) (5 mg / kg / day and 10 mg / kg / day), non-adverse treatment-related clinical signs such as muscle twitching, lethargy, swelling of the nose, chin, and / or cheeks, swelling of the forelimbs, reluctance to walk and / or stand, reduced activity, ataxia, and increased respiration were noted throughout the study at both dose levels, but were transient and resolved during the recovery period. There were no significant changes in rat body weights, except in the 20 mg / kg dose group, where a significant decrease in body weight was recorded in both male and female rats (data not shown). Food consumption was not significantly affected by treatment administration in rats at any dose level. Due to an unexpected death and treatment-related adverse clinical findings at 20 mg / kg, the study in this dose group was terminated early, and the remaining rats were euthanized prior to their scheduled administration on days 1 or 2. Adverse clinical findings associated with mortality included coldness to the touch, recumbency, abnormal coloration, lameness, severe dehydration, shivering, and labored breathing.

[0048] No changes in hematological parameters related to treatment with 5 mg / kg were observed in male and female rats at the end of treatment (day 8) (Table 5). RBC volume distribution width (RDW) was significantly elevated at the end of recovery in female rats at 5 mg / kg and was outside the historical control range (HCR) for female Sprague Dawley rats (data not shown). At 10 mg / kg, the absolute number of reticulocytes in male rats treated with 10 mg / kg was significantly decreased at the end of treatment. However, this value was within the HCR range for male Sprague Dawley rats of this age and was therefore not considered to be adverse (18). In female rats, WBC, as well as absolute lymphocyte and monocyte counts were significantly increased at the end of treatment when compared with controls. However, these increases were not considered to be adverse due to the lack of dose-dependency and the reversibility of the changes by the end of the recovery period (data not shown). At the end of recovery, mean cellular hemoglobin (MCH) and mean cellular hemoglobin concentration (MCHC) were moderately increased in male rats compared to controls, but the values ​​remained within the reference range for male rats of similar age. At 20 mg / kg, white blood cell counts (WBC), relative and absolute neutrophil counts, relative and absolute monocyte counts, and relative and absolute large unclassified cell (LUC) counts were significantly elevated in male rats, whereas relative lymphocyte counts, relative and absolute eosinophil counts, and platelet counts were significantly decreased on day 2 compared to controls at the end of treatment. The values ​​of relative and absolute neutrophil counts, relative lymphocyte counts, and relative and absolute monocyte counts were outside the HCR range for male rats. In female rats, there was a significant increase in absolute reticulocyte and monocyte counts when compared to controls at the end of treatment, whereas a significant decrease in relative and absolute eosinophil counts was observed on day 2 (interim euthanasia). Due to early termination of the study in the 20 mg / kg group, the reversibility of changes in these parameters could not be determined. However, despite statistical significance, these changes in female rats were not considered adverse as the values ​​were within the HCR for female rats.

[0049] [Table 5]

[0050] There were no significant treatment-induced abnormalities in serum biochemical parameters at the end of treatment in male and female rats administered 5 mg / kg / day or 10 mg / kg / day (Table 6). At the end of recovery, glucose levels were slightly elevated in female rats at 10 mg / kg (data not shown). At the highest dose examined (20 mg / kg), chloride and albumin levels were significantly decreased, whereas serum urea nitrogen (Urea N) levels were increased in male rats on day 2 compared to controls. In female rats, alanine aminotransferase (ALT), calcium, and gamma-glutamyltransferase (GGT) levels were significantly elevated compared to controls. In both male and female rats, albumin / globulin ratio (A / G), creatinine, glucose, and triglyceride levels were significantly elevated, whereas total protein (TP), sodium (Na), and globulin levels were significantly decreased, when compared to controls. Albumin, A / G, globulin, urea N, TP, and triglyceride levels were outside the HCR range in male rats, and A / G, GGT, globulin, glucose, TP, and triglyceride were outside the HCR range in female rats. The remaining parameters were within the HCR range and therefore considered not to be harmful.

[0051] [Table 6]

[0052] There were no treatment-related changes in coagulation parameters in male and female rats in the 5 mg / kg or 10 mg / kg groups at the end of treatment (Table S3) or at the end of recovery (data not shown). However, at the end of treatment, prothrombin time (PT) and activated partial thromboplastin time (APTT) were significantly elevated in both male and female rats at 20 mg / kg, whereas fibrinogen levels were decreased in male rats, when compared with controls. Of these, fibrinogen levels were outside the HCR range in male rats, and both PT and APTT were outside the HCR range in female rats.

[0053] [Table 7]

[0054] There were no treatment-related changes in urinalysis parameters from male and female rats dosed at 5 mg / kg or 10 mg / kg (data not shown). Due to early death, overnight urine samples were not collected in rats in the 20 mg / kg group and therefore could not be evaluated. No treatment-related abnormalities were detected during post-exposure ophthalmologic evaluation in either dose group. Treatment-related effects on the injection site could not be evaluated in this study due to surgical catheterization. Histopathological evaluation of rats in the 20 mg / kg group showed discoloration in the kidneys, brain, and lungs, and the bronchi and trachea were filled with fluid at the time of interim euthanasia. There were no macroscopic findings related to RTD-1 (SEQ ID NO: 1) treatment in either dose group at interim euthanasia (20 mg / kg group, day 2), terminal euthanasia (day 8), or recovery euthanasia (day 24). Significant treatment-related microscopic findings included a non-detrimental dose-dependent increased incidence of minimal to mild hepatic necrosis at the end of treatment in female rats dosed at 5 mg / kg / day and 10 mg / kg / day. However, hepatic necrosis was not seen in euthanized female animals (5 mg / kg / day and 10 mg / kg / day) at the end of the recovery period and did not result in increases in parameters included in the liver function panel (TP, albumin, total bilirubin, and liver enzymes). These findings were therefore considered reversible and not detrimental. Treatment-related adverse severity ranging from minimal to mild hepatic necrosis, defined by focal to multifocal areas of lytic to coagulative necrosis, was observed in male rats dosed at 20 mg / kg. Mild to severe adrenal necrosis, characterized by unilateral to bilateral coagulative cortical to corticomedullary necrosis, was observed in both male and female rats dosed at 20 mg / kg.

[0055] Based on the lack of adverse clinical signs or abnormalities in parameters at the 10 mg / kg dose, a no observed adverse effect level (NOAEL) was established in rats at 10 mg / kg / day. The lowest observed adverse effect level (LOAEL), defined by the FDA as the lowest dose with adverse effects tested in preclinical species, was established at 20 mg / kg in rats (19).

[0056] Cynomolgus monkey study: Overall, RTD-1 (SEQ ID NO: 1) at doses up to 15 mg / kg was well tolerated in male and female monkeys. In the non-GLP dose range-finding study, all animals survived the study without any treatment-related adverse clinical signs. Based on these data, the maximum tolerated dose (MTD) was set at 15 mg / kg. Similarly, in the GLP-compliant 10-day repeat-dose TK study, no deaths or unscheduled euthanasias occurred at any dose level. Furthermore, no significant changes in body weight were observed in monkeys. A treatment-related decrease in food consumption was observed in females at 10 mg / kg / day and in both sexes at 15 mg / kg / day during the dosing period, but this was not considered adverse as the animals had recovered to baseline by the end of recovery and the changes in food consumption were not linked to changes in body weight or adverse clinical findings.

[0057] Treatment-related non-adverse hematological changes at the end of treatment included a statistically significant but moderate increase in absolute LUC counts in males and absolute monocyte counts in females at 15 mg / kg compared to their respective controls (data not shown). However, the increase in absolute LUC counts was not considered adverse as it had resolved by the end of recovery (data not shown), and the increase in absolute monocyte counts observed in females was within the range of the HCR for female cynomolgus monkeys (20). The most notable non-adverse but significant treatment-related changes in serum biochemistry parameters included a slight decrease in inorganic phosphorus (Phos) levels and an increase in glucose levels at the end of treatment in females at 15 mg / kg / day. However, these changes were not considered adverse despite statistical significance as the magnitude of the changes was small, and the elevated values ​​were still within the range of the HCR for female cynomolgus monkeys. A trend towards increased fibrinogen levels was noted in all treated monkeys when compared to the control group and baseline levels, but this change did not reach statistical significance and returned to baseline by the end of the recovery period (data not shown). There were no treatment-related changes in urinalysis parameters from cynomolgus monkeys at any dose level, or abnormalities in ophthalmological evaluation or electrocardiogram after exposure (data not shown). The lack of significant changes in clinical pathology parameters in any of the animals with doses up to 15 mg / kg supports the results from the non-GLP dose-ranging study in cynomolgus monkeys, and the MTD of 15 mg / kg was also established based on the lack of adverse effects on mortality, clinical findings, or body weight with intravenous treatment with RTD-1 (SEQ ID NO: 1) up to 15 mg / kg. Due to the small number of measurements taken at the end of the recovery period, statistical analysis of clinical pathology parameters at this time point could not be performed.

[0058] Treatment-related macroscopic findings were noted at the injection site in three 15 mg / kg animals, including abrasions and abnormal texture likely due to repeated catheter insertions; however, there were no treatment-related macroscopic findings at the end of treatment or recovery. In the main group, microscopic evaluation revealed treatment-related thrombosis at the injection site at the end of treatment, but there was no dose trend in incidence and / or severity. In the recovery group, treatment-related thrombosis, acute inflammation, edema, hemorrhage, and fibrosis at the injection site were noted in animals dosed with 15 mg / kg at the end of recovery. However, comparable findings were noted at the injection site in control animals in the recovery group, including minimal to mild injection site fibrosis and mild chronic thrombosis, suggesting that these findings were treatment-related. Finally, minimal to mild intravascular thrombosis (thromboembolism) was observed at the end of treatment (day 11) in the lungs of only treated animals (males receiving doses of 10 mg / kg / day or higher and females receiving doses of 5 mg / kg / day or higher). The pulmonary thrombi contained variable numbers of inflammatory cells both within the thrombus and in the surrounding connective tissue, and were primarily located in the mid to small arteries and capillaries of the alveolar wall in two animals. It was concluded that the thromboembolism was related to peptide administration based on the composition of the thrombi found in the lungs, but that the thrombi were likely emboli from thrombi formed at the injection site. These findings also resolved by the end of the recovery period, as no thrombosis was identified in any lung sections in control or 15 mg / kg-treated monkeys.

[0059] A summary of the NOAEL and LOAEL determined in each species is listed in Table 8. Considering that repeated doses of RTD-1 (SEQ ID NO: 1) up to 15 mg / kg / day were well tolerated in both sexes, the NOAEL was set at 15 mg / kg / day in cynomolgus monkeys. The highest dose tested in the GLP study was well tolerated, so a LOAEL could not be determined in cynomolgus monkeys. Based on this analysis, the HED equivalent to the NOAEL and LOAEL in rats is 3.1 mg / kg and 11.7 mg / kg for 70 kg individuals, and the HED equivalent to the NOAEL in cynomolgus monkeys is 15.9 mg / kg for 70 kg individuals.

[0060] [Table 8]

[0061] safety Male and female Sprague Dawley rats (n=16-21 / sex) were evaluated for potential toxicity and reversibility of any findings following repeated dosing of RTD-1 (SEQ ID NO: 1) for 7 days. Rats were divided into three subgroups within each dosing group: primary (n=10 / sex), recovery (n=0-5 / sex), and toxicokinetics (TK) (n=3-6 / sex).

[0062] Mortality and Clinical Observations: Detailed clinical observations were recorded in all rats, starting one week prior to study initiation and weekly throughout the study, including the day of necropsy. Detailed clinical observations were recorded in cynomolgus monkeys, once prior to dosing and weekly after study initiation, including the day of necropsy. All animals were observed / monitored for mortality twice daily from the time of arrival until the time of death.

[0063] Body weight and food consumption: Individual body weights were recorded once before dosing in all rats and once every week after dosing initiation in cynomolgus monkeys three times before dosing and at least once every week after dosing initiation. Food consumption was quantitatively measured per cage once a week starting on day 1 throughout the study in rats and assessed once a day throughout the study in cynomolgus monkeys.

[0064] Hematology, blood chemistry and coagulation: Blood samples for hematology, coagulation and clinical chemistry were collected from the retro-orbital sinus on scheduled days (days 8, 24 and 25 for rats belonging to main, recovery and TK groups, respectively) or at unscheduled necropsy. Blood samples were collected by venipuncture in cynomolgus monkeys before dosing and at the end of treatment (day 11) and at the end of recovery in a small subset of male and female monkeys in the placebo (n=2 / sex) and 15 mg / kg groups (n=2-3 / sex).

[0065] Urinalysis: Overnight urine samples were collected in rats prior to euthanasia and in cynomolgus monkeys prior to dosing, on day 10, and at the end of recovery. Ophthalmology: Ophthalmologic examinations consisting of fundus examination (indirect ophthalmoscopy) and biomicroscopy (slit lamp) were performed in rats before dosing and on day 6, and in cynomolgus monkeys before dosing and on day 8.

[0066] Electrocardiogram (ECG): In cynomolgus monkeys, ECGs were collected pre-dose, within 10 minutes after the end of the infusion on day 8, and 2 days prior to necropsy. method The pharmacokinetics and safety of intravenous (iv) injection of RTD-1 (SEQ ID NO: 1) was studied in mice, rats, cynomolgus monkeys, and vervet monkeys. Lyophilized RTD-1 (SEQ ID NO: 1) (purity >98%) was dissolved in filter-sterilized saline and used for injection in mice and one vervet monkey. RTD-1 (SEQ ID NO: 1) solutions using rats and cynomolgus monkeys were prepared by diluting formulated RTD-1 (SEQ ID NO: 1) (12.5 mg / ml in 1% propylene glycol and 20 mM sodium acetate, pH 6.5) with filter-sterilized saline. A summary of the study design and safety evaluation schedule is listed in Tables 9 and 10, respectively. The study included single and repeated dose-ranging experiments. All protocols were approved by the local IACUC before the start of the study.

[0067] [Table 9]

[0068] [Table 10]

[0069] Pharmacokinetics Mouse studies: All procedures and protocols involving the use of animals were reviewed and approved by the University of Southern California (USC) IACUC (protocol #20538). Briefly, a single iv bolus injection of 5 mg / kg of RTD-1 (SEQ ID NO: 1) was administered into the lateral tail vein of male (31.7 g-37.7 g) and female (25.2 g-35.6 g) CD-1 mice (Charles River Laboratories). A total of 24 mice were divided into six groups (n=2 / sex / group) and each group was assigned to one pre-determined time point. Blood samples were collected by terminal cardiac puncture at 0.25, 1, 2, 4, 8, and 24 hours after administration. Collected samples were centrifuged to separate plasma and stored at -80°C until analysis.

[0070] Rat Study: The pharmacokinetics of RTD-1 (SEQ ID NO: 1) in Sprague-Dawley rats was evaluated as part of a Good Lab Practice (GLP) 7-day toxicity study. The study protocol was reviewed and approved by Citoxlab USA IACUC and was performed in accordance with guidelines from the USA National Research Council. On the day of dosing (Day 1), male (229g-272g) and female (186g-214g) rats (n=6 / sex / group) were assigned to receive repeated doses of 0 (placebo), 5, 10 or 20 mg / kg RTD-1 (SEQ ID NO: 1) by iv infusion (20 min ± 3 min) once daily for 7 days. The intravenous route was chosen since it is the intended route of administration for prophylactic treatment of microbial infections. Doses were selected based on a pilot single-escalation dose study in rats that established an MTD of 20 mg / kg (data not shown). Two subgroups of rats (n=3 / sex / group) were assigned with an alternating blood sampling scheme: Subgroup A was bled at 0 hours (pre-dose), 0.5 hours, 6 hours, and 24 hours after infusion, and Subgroup B was bled at 0.083 hours, 2 hours, and 12 hours after infusion. The 24 hour post-infusion sample on day 1 was taken before administration of the dose on day 2. Serial blood samples were collected in K2EDTA tubes at the above time points on days 1 and 7, and once on day 25 (recovery). Samples were centrifuged at 2,700 g for 10 minutes at 5° C. to separate plasma from blood and stored at −80° C. until analysis.

[0071] Cynomolgus Monkey Study: A non-GLP dose range-finding PK study and a GLP 10-day toxicity study were conducted in cynomolgus monkeys (Macaca fascicularis). The study protocol was reviewed and approved by the Citoxlab USA IACUC and was conducted in accordance with guidelines from the USA National Research Council. The non-GLP dose range-finding study included a single ascending dose evaluation and a repeat dose evaluation. In the single ascending dose PK study, male (3.49 kg to 3.57 kg) and female (2.66 kg to 2.79 kg) cynomolgus monkeys (n=1 / sex / group) were randomly assigned to one of two dose groups. Group 1 received a single dose of 0.3 mg / kg RTD-1 (SEQ ID NO: 1) as an iv infusion (60 min ± 5 min) on day 1 and a single dose of 3 mg / kg RTD-1 (SEQ ID NO: 1) on day 3, while group 2 received a single dose of 1 mg / kg RTD-1 (SEQ ID NO: 1) on day 1 and a single dose of 10 mg / kg RTD-1 (SEQ ID NO: 1) on day 3. The doses used in this study were based on pilot studies in cynomolgus monkeys, which demonstrated the safety of a single iv dose of 10 mg / kg or less (data not shown). Blood samples were collected in K2EDTA tubes at the following time points: pre-dose, approximately 0.083 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, and 24 hours after the end of the infusion on days 1 and 3. In the repeat dose study, 15 mg / kg RTD-1 (SEQ ID NO: 1) was administered by iv infusion (60 min ± 5 min) once daily for 7 days (n=1 / sex). Serial blood samples were collected in K2EDTA pre-dose, and approximately 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours after the end of infusion on days 1, 4, and 7. Samples 24 hours after infusion on days 1 and 4 were taken prior to administration of the doses on days 2 and 5, respectively. In a GLP 10-day toxicity study, PK was evaluated after repeated ascending dose administration of RTD-1 (SEQ ID NO: 1).Male (2.52 kg-3.65 kg) and female (2.39 kg-3.18 kg) cynomolgus monkeys (n=3-5 / sex / group) were administered placebo (sterile saline) or 5, 10 or 15 mg / kg RTD-1 (SEQ ID NO: 1) by iv infusion (60 min±5 min) once daily for 10 days. Serial blood samples were collected in K2EDTA tubes at the following time points: pre-dose, 0.083, 0.5, 2, 6, and 12 hours post-infusion on day 1, and pre-dose, 0.083, 0.5, 2, 6, 12, and 24 hours post-infusion on day 10. Additional blood samples were taken on days 12 and 24 from monkeys (n=2 / sex / group) administered 0 or 15 mg / kg. All samples were centrifuged at approximately 2700 xg for 10 minutes at approximately 5°C to isolate plasma, which was then stored at -80°C until analysis.

[0072] Vervet Monkey Study: In a pilot safety study, a single dose of 3 mg / kg RTD-1 (SEQ ID NO: 1) was administered by iv bolus to adult male vervet / African green monkeys (Chlorocebus aethiops sabaeus). Serial blood samples were taken at 0.5, 1, 4, 8, 24, 48, 72, 96, 120, and 192 hours after dosing.

[0073] Biological Analysis: Clarified plasma samples from mice were diluted directly in 5% formic acid / 5% acetonitrile. Quantitative analysis of RTD-1 (SEQ ID NO:1) was performed by LC-MS / MS using reversed-phase liquid chromatography (XBridge BEH Phenyl 3.5 μm 3×100 mm column, Waters #186003328) on an Acquity H-Class UPLC (Waters) coupled to a Xevo TQ-S tandem electrospray mass spectrometry running MassLynx V4.1 (Waters). Quantitative mass spectrometry was performed by multiple reaction monitoring transition 417.32>517.21 with area under the curve determined by TargetLynx (Waters). A synthetic theta defensin-like peptide was used as an internal standard (IS). The lower limit of quantification (LLOQ) of the assay was 1 ng / mL. Intra- and inter-assay precision (percent coefficient of variation [CV]) was ≤3%, and intra- and interassay accuracy (% relative error) was ≤5%. Plasma RTD-1 (SEQ ID NO: 1) concentration analysis of rats and cynomolgus monkeys was performed at MicroConstants (San Diego, CA) by HPLC using a Mac-Mod Analytical ACE C4 column. The mobile phase was nebulized with heated nitrogen with a Z-Spray source / interface set in electrospray positive ionization mode, and compounds were detected using MS / MS (LLOQ=10.0 ng / mL). Details of the method are summarized in MicroConstants method no. MN20038. Clarified plasma samples from continuously sampled vervet monkeys were quantified by ultra-high performance liquid chromatography Waters Acquity H-Class UPLC. Plasma samples were directly diluted (1:10) in 5% formic acid / 5% acetonitrile and quantified by photodiode array (PDA AUC at 210 nm) using a (C18 XBridge BEH 2.5 μm 2.1 × 150 mm Waters #186006709) running Empower software.Peak and mass confirmation of RTD-1 (SEQ ID NO:1) was performed on the post-PDA eluate using a Micromass Quattro Ultima mass spectrometer with MassLynx 4.1 (Waters). The lower limit of quantification (LLOQ) ranged from 10-30 ng / mL (determined by sample background) to an upper limit of 50 ug / mL. RTD-2 (10 ug / mL) was used as an internal standard. Intra- and inter-assay precision (% coefficient of variation [CV]) was ≤3%, and intra- and inter-assay accuracy (% relative error) was ≤5%.

[0074] Pharmacokinetic Analysis: Noncompartmental analysis (NCA) was performed using Phoenix® WinNonlin (version 8.3.1, Certara USA, Inc.; Princeton, NJ) to determine PK parameters in mice, rats, cynomolgus monkeys, and vervet monkeys. Nominal sampling times were used for the analysis, and data below the lower limit of quantitation of the assay were excluded from the analysis. Maximum plasma concentrations (Cmax) were determined from visual inspection of the data. In addition, the following parameters were calculated: terminal phase elimination rate constant (λz), area under the curve extrapolated to infinity (AUC0-∞), area under the curve up to the dosing interval (AUCτ), mean residence time (MRT), clearance (CL), and volume of distribution at steady state (Vss). AUC was calculated using the linear up, log down method, and λz was calculated using up to the last four data points of the log-linear terminal phase of the concentration-time profile. Because the data were sparsely sampled in mice and rats, the Phoenix WinNonlin sparse sampling calculation method was used, which produced a single estimate with no standard error for all parameters except Cmax. For rats and cynomolgus monkeys, all parameters were calculated using sampling times from the start of iv infusion.

[0075] Dose proportionality: Dose proportionality of Cmax and AUC0-∞ was assessed in cynomolgus monkeys administered single iv doses of RTD-1 (SEQ ID NO: 1) ranging from 0.3 to 15 mg / kg using a natural log-transformed power model (37). Dose proportionality was concluded when the slope of the linear regression and the corresponding 95% confidence intervals were included.

[0076] Interspecies allometric scaling: Single-dose PK data from mice, rats, cynomolgus monkeys, and vervet monkeys were used to predict human PK parameters using simple allometry. The relationship between CL or Vss derived from the NCA and body weight was described using the following equation: Y=a·BW b where Y is the PK parameter (e.g., CL or Vss), BW is the body weight of the species, a is the allometric coefficient, and b is the allometric exponent (34, 38). Linear regression was performed on the log-transformed data. The predicted human equivalent dose was calculated based on the allometrically scaled clearance using the following formula: Dose = CL * AUC 0-∞ Corresponding mean AUCτ and AUC at NOAEL and LOAEL 0-∞ were used to convert doses at the NOAEL and LOAEL in preclinical animals to HEDs, respectively.

[0077] In female rats 14 Biodistribution of [C]-radiolabeled RTD-1 (SEQ ID NO: 1). Approximately 4 million CPM of [C] was administered to five SD rats (body weight 195 g and 200 g) equipped with a jugular vein catheter (JVC), respectively. 14 200 μL of 5 mg / mL RTD-1 (SEQ ID NO: 1) in saline containing [C]-RTD-1 (SEQ ID NO: 1) was injected. 14 C-RTD-1 (SEQ ID NO: 1) was synthesized by replacing the natural glycine residue at position 1 of the cyclic peptide with 14C-labeled glycine was substituted with 100 μL saline. The JVC line was flushed with 70% isopropyl alcohol and the line plug was removed. A new 25G blunt needle with a 1mL syringe containing the injection solution was used for each injection. The JVC line was first flushed with 100 μL saline, followed by RTD-1 (SEQ ID NO: 1) solution, then with an additional 100 μL saline. Tissues and organs were collected in separate vials and weighed. Small organs such as lymph nodes, kidneys, and heart / lungs were processed whole. Larger organs (e.g., liver, muscle, subcutaneous fat pads) were sampled from representative areas or areas of interest (e.g., subcutaneous tissue at the injection site). Organs were then dissolved in 2 mL of SOLVABLE (Perkin Elmer 6NE9100) for up to 1 g of tissue. For skin and most of the other organs, 4 mL or 6 mL were added. The vials were incubated in a 60°C water bath for 18-22 hours, then removed and cooled to room temperature. 2 mL of lysed tissue was added to a new scintillation tube along with 100 μL of 0.1 M EDTA and 2×100 μL of 30% hydrogen peroxide for color compensation. Samples were left at room temperature for 1 hour, incubated in a 37°C incubator for 1 hour, and then incubated in a 60°C water bath for 1 hour. If necessary to prevent boiling, samples were briefly removed from the heat source before continuing. After the samples had cooled, 10 mL of Ultima Gold (Perkin Elmer 6013321) was added to each vial, and the contents were then mixed and left in the dark at 22°C for 1 hour. Scintillation counts were the average of two counts per minute using a Packard TRI-CARD 2100 TR scintillation counter. Urine was collected for 1 hour or 24 hours after iv injection, and 500 μL was added to scintillation vials processed as above for scintillation counting. The stomach and its contents were lysed with 6 mL of SOLVABLE and processed in the same manner as the other tissues. The duodenum, jejunum, and ileum were flushed with saline to remove luminal contents, and the tissues were then processed with SOLVABLE as above.The colon was opened lengthwise to remove fecal contents, rinsed with saline to remove remaining luminal contents, and then processed as above. Feces were collected, transferred to a 500 mL plastic cup, treated with 50 mL of 7% sodium hypochlorite, and incubated at 22°C for 1 h followed by 1 h in a 60°C water bath. 2 mL of the resulting suspension was transferred to a scintillation vial and mixed with 10 mL of scintillation fluid. Contents were mixed and allowed to sit for 1 h before scintillation counting.

[0078] safety Safety assessments in rats and cynomolgus monkeys were based on clinical findings, survival, body weight, food consumption, clinical pathology (hematology, clinical chemistry, and coagulation), urinalysis, ophthalmology, gross findings at necropsy, and microscopic histopathology. The assessment schedules for these studies are summarized in Table 2.

[0079] statistical analysis Statistical analysis of PK data was performed using GraphPad Prism version 9.1.2 (GraphPad Software, Inc., San Diego, CA). Shapiro-Wilk test was used to check for normality. One-way ANOVA with Bonferroni's multiple comparison test was performed to compare Cmax between doses (5, 10 and 20 mg / kg) in rats, and λz, AUC, Cmax, CL and Vss between doses (5, 10 and 15 mg / kg) in cynomolgus monkeys. Unpaired t-tests were used to compare PK parameters (λz, AUC, Cmax, CL and Vss) between different days (day 1 vs. day 10) and between genders within each dose group. Statistical analysis of safety data was performed using SAS 9.4, considering 95% statistical significance. Differences in body weight, body weight change and clinical pathology (hematology, serum chemistry, coagulation) were compared between each dose group using Kruskal Wallis and Dunn's multiple comparison test. Data analyses were performed independently for each sex.

[0080] It will be apparent to those skilled in the art that many more modifications are possible in addition to those already described without departing from the spirit of the invention herein. Thus, the subject matter of the present invention should not be limited except to the spirit of the appended claims. Moreover, 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 "comprises" and "comprising" should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that a referenced element, component, or step may be present, utilized, or combined with other elements, components, or steps not expressly referenced. When a claim herein refers to at least one of something selected from the group consisting of A, B, C...N, the text should be interpreted as requiring only one element from that group, not A+N, or B+N, etc.

[0081] [Table 11-1]

[0082] [Table 11-2]

[0083] [Table 11-3]

[0084] [Table 11-4]

[0085] [Table 11-5]

[0086]

Table 11-6

[0087]

Table 11-7

[0088]

Table 11-8

[0089]

Table 11-9

[0090]

Table 11-10

[0091]

Table 11-11

[0092]

Table 11-12

[0093]

Table 11-13

[0094]

Table 11-14

[0095]

Table 11-15

[0096]

Table 11-16

[0097]

Table 11-17

[0098]

Table 11-18

[0099]

Table 11-19

[0100]

Table 11-20

Claims

1. 1. A method of providing prophylactic treatment against infection by a microbial organism, comprising: identifying an individual in need of prophylactic treatment against said microbial infection; A method comprising administering to the individual in need of treatment an effective amount of an effective θ-defensin or an effective θ-defensin analog, wherein the effective amount provides antibacterial activity against the microorganism at least 50% of the peak plasma concentration of the effective θ-defensin or the effective θ-defensin analog.

2. The method of claim 1, further comprising identifying an effective θ-defensin or an effective θ-defensin analog that maintains at least 50% of the peak plasma concentration of the effective θ-defensin or effective θ-defensin analog for at least 4 hours after administration.

3. 3. The method of claim 1 or 2, wherein the effective θ-defensin is selected from the group consisting of RTD-1 (SEQ ID NO:1), RTD-2 (SEQ ID NO:2), RTD-3 (SEQ ID NO:3), RTD-4 (SEQ ID NO:4), RTD-5 (SEQ ID NO:5), RTD-6 (SEQ ID NO:6), BTD-1 (SEQ ID NO:7), BTD-2 (SEQ ID NO:8), BTD-3 (SEQ ID NO:9), BTD-4 (SEQ ID NO:10), BTD-5 (SEQ ID NO:11), BTD-6 (SEQ ID NO:12), BTD-7 (SEQ ID NO:13), BTD-8 (SEQ ID NO:14), BTD-9 (SEQ ID NO:15), and / or BTD-10 (SEQ ID NO:16), and a peptide derived from HTDp (SEQ ID NO:17).

4. The method of claim 1 or claim 2, wherein the effective θ-defensin analogue is selected from the group consisting of peptide 1 (SEQ ID NO:18), peptide 2 (SEQ ID NO:19), peptide 3 (SEQ ID NO:20), peptide 4 (SEQ ID NO:21), peptide 5 (SEQ ID NO:22), peptide 6 (SEQ ID NO:23), peptide 7 (SEQ ID NO:24), peptide 8 (SEQ ID NO:25), peptide 9 (SEQ ID NO:26), peptide 10 (SEQ ID NO:27), peptide 11 (SEQ ID NO:28), peptide 12 (SEQ ID NO:29), peptide 13 (SEQ ID NO:30), peptide 14 (SEQ ID NO:31), and peptide 15 (SEQ ID NO:32).

5. 5. The method of claim 1, wherein the individual is further identified as a high-risk individual.

6. 6. The method of claim 5, wherein the high risk individuals are identified as: newborns, premature infants, children with underdeveloped or partially developed immune systems, elderly individuals, individuals recovering from surgery, individuals recovering from accidental injury, individuals with cancer, immunocompromised individuals, individuals who have undergone organ, bone marrow, or stem cell transplants, individuals undergoing or recovering from chemotherapy, individuals undergoing or recovering from radiation therapy, individuals undergoing or recovering from immunotherapy, individuals undergoing or recovering from immunosuppressive therapy, individuals with underlying heart disease and / or cardiac damage, individuals with underlying renal disease and / or renal damage, individuals with underlying liver disease and / or liver damage, individuals with underlying lung disease and / or lung damage, individuals with obesity, etc.

7. 6. The method of claim 5, wherein the high-risk individuals are identified based on exposure to others within a distance that may transmit the microorganism.

8. The method according to any one of claims 1 to 7, wherein the microorganism is a virus.

9. The method of claim 6, wherein the virus is SARS-CoV-2.

10. The method of any one of claims 1 to 7, wherein the microorganism is a bacterium.

11. The method according to any one of claims 1 to 7, wherein the microorganism is a fungus.

12. The method of any one of claims 1 to 7, wherein the microorganism is a protozoan.

13. The method of any one of claims 1 to 12, wherein the effective θ-defensin is RTD-1 (SEQ ID NO: 1).

14. The method of any one of claims 1 to 13, wherein the effective θ-defensin or effective θ-defensin analog is administered at up to 15 mg / kg.

15. The method of any one of claims 1 to 14, wherein administration comprises a single administration of the effective θ-defensin or the effective θ-defensin analog.

16. 16. The method of claim 15, wherein the single dose comprises an amount of the effective θ-defensin or the effective θ-defensin analog to provide 0.1 mg / kg to 15 mg / kg to the individual in need of treatment.

17. The method of any one of claims 1 to 14, wherein administering comprises repeated administration of the effective θ-defensin or the effective θ-defensin analog.

18. 20. The method of claim 17, wherein the protocol comprises a dosing frequency of from once every 12 hours to once every 48 hours.

19. 19. The method of claim 17 or 18, wherein each administration comprises an amount of the effective θ-defensin or the effective θ-defensin analog sufficient to provide 0.1 mg / kg to 15 mg / kg to the individual in need of treatment.

20. The method of any one of claims 17 to 19, wherein the repeated administration comprises administration of a first amount of the effective θ-defensin or the effective θ-defensin analog and administration of a second amount of the effective θ-defensin or the effective θ-defensin analog.

21. Use of a formulation comprising a theta-defensin or a theta-defensin analog for prophylactic treatment against infection by a microorganism, the formulation comprising an effective theta-defensin or an effective theta-defensin analog identified as maintaining at least 50% of the peak plasma concentration of the effective theta-defensin or effective theta-defensin analog for at least four hours after administration, the formulation providing a sufficient amount of the effective theta-defensin or effective theta-defensin analog to provide an antibacterial effect for at least four hours.

22. 22. The use according to claim 21, wherein the effective θ-defensin is selected from the group consisting of RTD-1 (SEQ ID NO:1), RTD-2 (SEQ ID NO:2), RTD-3 (SEQ ID NO:3), RTD-4 (SEQ ID NO:4), RTD-5 (SEQ ID NO:5), RTD-6 (SEQ ID NO:6), BTD-1 (SEQ ID NO:7), BTD-2 (SEQ ID NO:8), BTD-3 (SEQ ID NO:9), BTD-4 (SEQ ID NO:10), BTD-5 (SEQ ID NO:11), BTD-6 (SEQ ID NO:12), BTD-7 (SEQ ID NO:13), BTD-8 (SEQ ID NO:14), BTD-9 (SEQ ID NO:15), and / or BTD-10 (SEQ ID NO:16), and a peptide derived from HTDp (SEQ ID NO:17).

23. The use of claim 21, wherein the effective θ-defensin analogue is selected from the group consisting of peptide 1 (SEQ ID NO:18), peptide 2 (SEQ ID NO:19), peptide 3 (SEQ ID NO:20), peptide 4 (SEQ ID NO:21), peptide 5 (SEQ ID NO:22), peptide 6 (SEQ ID NO:23), peptide 7 (SEQ ID NO:24), peptide 8 (SEQ ID NO:25), peptide 9 (SEQ ID NO:26), peptide 10 (SEQ ID NO:27), peptide 11 (SEQ ID NO:28), peptide 12 (SEQ ID NO:29), peptide 13 (SEQ ID NO:30), peptide 14 (SEQ ID NO:31), and peptide 15 (SEQ ID NO:32).

24. 24. The use according to any one of claims 21 to 23, wherein the microorganism is a virus.

25. The use according to claim 24, wherein the virus is SARS-CoV-2.

26. 24. The use according to any one of claims 21 to 23, wherein the microorganism is a bacterium.

27. 24. The use according to any one of claims 21 to 23, wherein the microorganism is a fungus.

28. 24. The use according to any one of claims 21 to 23, wherein the microorganism is a protozoan.

29. The use according to any one of claims 21 to 28, wherein the effective θ-defensin is RTD-1 (SEQ ID NO: 1).

30. 30. The use of any one of claims 21 to 29, wherein the formulation provides the effective θ-defensin or the effective θ-defensin analog at up to 15 mg / kg.

31. The use of any one of claims 21 to 30, wherein the formulation is formulated to provide prophylaxis after a single administration of the effective theta-defensin or the effective theta-defensin analogue.

32. The use of any one of claims 21 to 30, wherein the formulation is formulated to provide prophylaxis following repeated administration of the effective θ-defensin or the effective θ-defensin analogue.

33. The use of claim 32, wherein the formulation is formulated to provide an amount of the effective θ-defensin or the effective θ-defensin analog sufficient to provide 0.1 mg / kg to 15 mg / kg in each of the repeated administrations.

34. A formulation for providing prophylactic treatment of infection by a microorganism, comprising a theta-defensin or a theta-defensin analog for prophylactic treatment against infection by a microorganism, said formulation comprising an effective theta-defensin or an effective theta-defensin analog identified as maintaining at least 50% of the peak plasma concentration of the effective theta-defensin or effective theta-defensin analog for at least four hours after administration, said formulation providing an amount of the effective theta-defensin or effective theta-defensin analog sufficient to provide an antibacterial effect for at least four hours.

35. 35. The formulation of claim 34, wherein the effective θ-defensin is selected from the group consisting of RTD-1 (SEQ ID NO:1), RTD-2 (SEQ ID NO:2), RTD-3 (SEQ ID NO:3), RTD-4 (SEQ ID NO:4), RTD-5 (SEQ ID NO:5), RTD-6 (SEQ ID NO:6), BTD-1 (SEQ ID NO:7), BTD-2 (SEQ ID NO:8), BTD-3 (SEQ ID NO:9), BTD-4 (SEQ ID NO:10), BTD-5 (SEQ ID NO:11), BTD-6 (SEQ ID NO:12), BTD-7 (SEQ ID NO:13), BTD-8 (SEQ ID NO:14), BTD-9 (SEQ ID NO:15), and / or BTD-10 (SEQ ID NO:16), and a peptide derived from HTDp (SEQ ID NO:17).

36. 35. The use of the formulation of claim 34, wherein the effective θ-defensin analogue is selected from the group consisting of peptide 1 (SEQ ID NO:18), peptide 2 (SEQ ID NO:19), peptide 3 (SEQ ID NO:20), peptide 4 (SEQ ID NO:34), peptide 5 (SEQ ID NO:22), peptide 6 (SEQ ID NO:23), peptide 7 (SEQ ID NO:24), peptide 8 (SEQ ID NO:25), peptide 9 (SEQ ID NO:26), peptide 10 (SEQ ID NO:27), peptide 11 (SEQ ID NO:28), peptide 12 (SEQ ID NO:29), peptide 13 (SEQ ID NO:30), peptide 14 (SEQ ID NO:31), and peptide 15 (SEQ ID NO:32).

37. 37. The formulation of any one of claims 34 to 36, wherein the microorganism is a virus.

38. 38. The formulation of claim 37, wherein the virus is SARS-CoV-2.

39. 37. The formulation of any one of claims 34 to 36, wherein the microorganism is a bacterium.

40. 37. The formulation of any one of claims 34 to 36, wherein the microorganism is a fungus.

41. 37. The formulation of any one of claims 34 to 36, wherein the microorganism is a protozoan.

42. The formulation of any one of claims 34 to 41, wherein the effective θ-defensin is RTD-1 (SEQ ID NO: 1).

43. 43. The formulation of any one of claims 34 to 42, wherein the formulation is formulated to provide up to 15 mg / kg of the effective θ-defensin or the effective θ-defensin analogue.

44. 44. The formulation of any one of claims 34 to 43, wherein the formulation is formulated to provide prophylaxis after a single administration of the effective theta-defensin or the effective theta-defensin analog.

45. 44. The formulation of any one of claims 34 to 43, wherein the formulation is formulated for prophylaxis following repeated administration of the effective theta-defensin or effective theta-defensin analog.

46. The formulation of claim 45, wherein the formulation is formulated to provide an amount of the effective θ-defensin or the effective θ-defensin analog sufficient to provide 0.1 mg / kg to 15 mg / kg in each of the repeated administrations.

47. Use of a θ-defensin or a θ-defensin analog in the preparation of a formulation for providing prophylactic treatment against infection by a microorganism, comprising a θ-defensin or a θ-defensin analog for prophylactic treatment against infection by a microorganism, wherein the formulation comprises an effective θ-defensin or an effective θ-defensin analog identified as maintaining at least 50% of the peak plasma concentration of the effective θ-defensin or effective θ-defensin analog for at least 4 hours after administration, and the formulation provides a sufficient amount of the effective θ-defensin or effective θ-defensin analog to provide an antibacterial effect for at least 4 hours.

48. 48. The use according to claim 47, wherein the effective θ-defensin is selected from the group consisting of RTD-1 (SEQ ID NO:1), RTD-2 (SEQ ID NO:2), RTD-3 (SEQ ID NO:3), RTD-4 (SEQ ID NO:4), RTD-5 (SEQ ID NO:5), RTD-6 (SEQ ID NO:6), BTD-1 (SEQ ID NO:7), BTD-2 (SEQ ID NO:8), BTD-3 (SEQ ID NO:9), BTD-4 (SEQ ID NO:10), BTD-5 (SEQ ID NO:11), BTD-6 (SEQ ID NO:12), BTD-7 (SEQ ID NO:13), BTD-8 (SEQ ID NO:14), BTD-9 (SEQ ID NO:15), and / or BTD-10 (SEQ ID NO:16), and a peptide derived from HTDp (SEQ ID NO:17).

49. The use of claim 47, wherein the effective θ-defensin analogue is selected from the group consisting of peptide 1 (SEQ ID NO:18), peptide 2 (SEQ ID NO:19), peptide 3 (SEQ ID NO:20), peptide 4 (SEQ ID NO:47), peptide 5 (SEQ ID NO:22), peptide 6 (SEQ ID NO:23), peptide 7 (SEQ ID NO:24), peptide 8 (SEQ ID NO:25), peptide 9 (SEQ ID NO:26), peptide 10 (SEQ ID NO:27), peptide 11 (SEQ ID NO:28), peptide 12 (SEQ ID NO:29), peptide 13 (SEQ ID NO:30), peptide 14 (SEQ ID NO:31), and peptide 15 (SEQ ID NO:32).

50. 50. The use according to any one of claims 47 to 49, wherein the microorganism is a virus.

51. The use according to claim 50, wherein the virus is SARS-CoV-2.

52. 50. The use according to any one of claims 47 to 49, wherein the microorganism is a bacterium.

53. 50. The use according to any one of claims 47 to 49, wherein the microorganism is a fungus.

54. 50. The use according to any one of claims 47 to 49, wherein the microorganism is a protozoan.

55. The use according to any one of claims 47 to 54, wherein the effective θ-defensin is RTD-1 (SEQ ID NO: 1).

56. 56. The use of any one of claims 47 to 55, wherein the formulation is formulated to provide up to 15 mg / kg of the effective θ-defensin or the effective θ-defensin analogue.

57. 57. The use of any one of claims 46 to 56, wherein the formulation is formulated to provide prophylaxis after a single administration of the effective theta-defensin or the effective theta-defensin analogue.

58. 57. The use of any one of claims 47 to 56, wherein the formulation is formulated to provide prophylaxis following repeated administration of the effective theta-defensin or effective theta-defensin analog.

59. The use of claim 58, wherein the formulation is formulated to provide an amount of the effective θ-defensin or the effective θ-defensin analog sufficient to provide 0.1 mg / kg to 15 mg / kg in each of the repeated administrations.

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