Composition and method of ENT-testosterone for the treatment of bacterial infections

Abstract

This disclosure generally relates to compositions and methods involving the use of enantiomerized testosterone (Ent-testosterone). The disclosed Ent-testosterone can be used to inhibit bacterial quorum sensing. This disclosure also relates to further compositions and methods for treating subjects having bacterial infections or conditions complicated by bacterial colonization. In some embodiments, the compositions further include a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is petrolatum.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 552,459, filed on 12 February 2024 and titled “COMPOSITIONS AND METHODS OF ENT-TESTOSTERONE FOR TREATMENT OF BACTERIAL INFECTIONS,” and to U.S. Provisional Patent Application No. 63 / 580,813, filed on 6 September 2023 and titled “COMPOSITIONS AND METHODS OF ENT-TESTOSTERONE FOR TREATMENT OF BACTERIAL INFECTIONS,” which are incorporated herein by reference in their entirety.

[0002] Incorporation of sequence lists This application includes a sequence listing submitted via the Patent Center in .XML format, which is incorporated herein by reference in its entirety. The WIPO sequence listing was created on 21 August 2024, and the XML copy is named 106546-816096-4247_Sequence listing.xml and is 19 kilobytes in size. [Background technology]

[0003] background 1. Field

[0004] The present invention relates to enantiomer testosterone (ENT-testosterone), as well as compositions and methods for using ENT-testosterone, for the treatment of bacterial infections.

[0005] 2.Background

[0006] Skin and soft tissue infections caused by Staphylococcus aureus pose a significant public health threat. Seven to ten percent of hospitalized patients suffer from skin and soft tissue infections, making them the third leading reason for emergency room visits. 75 percent of these skin infections are caused by Staphylococcus aureus. In addition to the disease burden caused by these pathogens, there is the emergence of antibiotic-resistant bacterial strains, such as methicillin-resistant Staphylococcus aureus (MRSA). MRSA is the most frequent cause of skin and soft tissue infections in emergency room settings and a major cause of hospital-acquired infections. Furthermore, MRSA infections have an overall social cost exceeding $1 billion annually.

[0007] Staphylococcus aureus is an opportunistic pathogen, capable of both quiescent colonization and lethal infection. Staphylococcus aureus encodes numerous virulence factors, from cell surface adherens to extracellular matrix-degrading enzymes, which enable it to bind to, invade, damage, and infect epithelial cells. The best-described system of virulence regulation is the accessory gene regulator (agr) quorum sensing system. Agr senses the concentration of autoinducible peptide (AIP) signals produced by Staphylococcus aureus or other bacteria in the environment. AIP is synthesized in a stepwise process. First, the propeptide AgrD is produced in the cytoplasm. The AgrD propeptide is then processed into AIP and secreted into the extracellular space by the intrinsic membrane endopeptidase AgrB. Studies have also shown the involvement of MroQ in the AIP biosynthesis pathway. When the bacteria are at a sufficient density and the concentration of AIP is sufficiently high ("quorum"), AIP binds to and is activated by AgrC histidine kinase. Upon activation, AgrC phosphorylates the response regulator AgrA, then autoinduces the expression of the Agr mechanism, which in turn induces the expression of RNAIII transcripts. RNAIII is the system's major effector, triggering increased production of toxins and exoenzymes in Staphylococcus aureus. In addition to responding to AIP, AgrC can respond to other environmental conditions, and recent studies have demonstrated that nitric oxide can inhibit Staphylococcus aureus via direct inhibition of AgrC. However, host-derived factors can drive Staphylococcus aureus cutaneous infections in vivo and influence bacterial pathogenesis as well as the course of skin infections and diseases. For example, bacterial cutaneous infections by Staphylococcus aureus have limited treatment options, creating an unmet need for the development of therapeutic agents to treat skin and soft tissue infections. [Overview of the project] [Means for solving the problem]

[0008] overview In one embodiment, the disclosure encompasses a composition for the treatment of bacterial infections in subjects requiring treatment for bacterial infections, the composition comprising enantiomer-testosterone (Ent-testosterone). In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is petrolatum.

[0009] In a further embodiment, a method for treating a subject having or at risk of having a bacterial infection is provided herein, the method comprising administering a composition comprising ent-testosterone to the subject.

[0010] This disclosure further encompasses methods for treating subjects who have or are at risk of having bacterial skin and soft tissue infections (SSTIs), the methods including administering a composition comprising ent-testosterone to the subjects.

[0011] In some embodiments, the disclosure includes methods for treating subjects having or at risk of having a condition complicated by the colonization of bacteria, the methods including administering a composition comprising ent-testosterone to the subjects. Further methods are provided herein for limiting tissue damage to subjects caused by bacterial pathogenicity-associated proteins, the methods including administering a composition comprising ent-testosterone to the subjects. In such embodiments, the tissue is skin tissue.

[0012] This disclosure further encompasses methods for reducing the pathogenicity of Staphylococcus species, which include contacting Staphylococcus species with a composition containing an effective amount of Ent-testosterone.

[0013] In some cases, the condition is atopic dermatitis, folliculitis, pemphigus, bullous pemphigoid, or cutaneous T-cell lymphoma.

[0014] In some embodiments, bacterial infections are caused by bacteria of the genus Staphylococcus. In some embodiments, bacterial infections are caused by Staphylococcus aureus. In some embodiments, Staphylococcus aureus includes methicillin-resistant Staphylococcus aureus (MRSA), methicillin-susceptible Staphylococcus aureus (MSSA), vancomycin-intermediate resistant Staphylococcus aureus (VISA), and vancomycin-resistant Staphylococcus aureus (VRSA). In some embodiments, Staphylococcus aureus includes Staphylococcus aureus strain LAC, Staphylococcus aureus strain USA 100, Staphylococcus aureus strain MW2, and Staphylococcus aureus strain MN EV. In other embodiments, bacterial infections are caused by Staphylococcus epidermidis.

[0015] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0016] In some embodiments, Ent-testosterone reduces the expression of pathogenicity factors or pathogenicity-associated protein genes by at least 10% compared to untreated controls. In some embodiments, the pathogenicity-associated protein gene is accessory gene regulator (agr) type I. In some embodiments, the pathogenicity-associated protein gene is agr type II. In some embodiments, the pathogenicity-associated protein gene is agr type III. In some embodiments, the pathogenicity-associated protein gene is agr type IV. In further embodiments, Ent-testosterone reduces staphylococcus-induced hemolysis by at least 10% and / or staphylococcus-induced neutrophil killing by at least 5%.

[0017] In some embodiments, ent-testosterone reduces bacterial quorum sensing by at least 10% compared to untreated controls. In some embodiments, the effective dose of ent-testosterone is approximately 10 nM to approximately 10 μM. In some embodiments, the effective dose of ent-testosterone reduces accessory gene regulator C histidine kinase (agrC).

[0018] Other technical features will be readily apparent to those skilled in the art from the following figures, descriptions, and claims. [Brief Description of the Drawings]

[0019] [Figure 1] Figure 1 is a schematic diagram of the coordinated expression of the agr system of virulence factors in Staphylococcus aureus.

[0020] [Figure 2] Figure 2 shows the relative expression of PSMα quantified by RT-PCR in Staphylococcus aureus strains LAC (type I), USA 100 (type II), MW2 (type III), and MN EV (type IV) treated with 10 nM Ent-testosterone, 10 nM testosterone, or vehicle. Mean ± SEM (error bars) are plotted. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns, not significant by unpaired two-tailed t-test.

[0021] [Figure 3] Figure 3 shows representative images of HSD3B6ΔSkin female mice transdermally infected with the bioluminescent MRSA agr-P3lux reporter strain of MRSA at 1 hour, 2 hours, 3 hours, and 4 hours after treatment with 10 nM Ent-testosterone, 10 nM testosterone, or vehicle. Female wild-type mice were transdermally infected with 1×10^6 CFU of the agr-P3 Staphylococcus aureus reporter and treated with testosterone or ent-T. Bioluminescence was quantified over time. 700 testosterone (n=4), ent-T (n=4), vehicle (n=4).

[0022] [Figure 4]Figure 4 shows the mean bioluminescence in HSD3B6ΔSkin female mice percutaneously infected with the bioluminescent MRSA agr-P3lux reporter strain of MRSA, 1 to 8 hours after treatment with 10 nM ent-testosterone, 10 nM testosterone, or vehicle. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.05, determined by unpaired t-tests.

[0023] [Figure 5] Figure 5 shows the time-dependent quantification of bioluminescence in the MRSAagr-P3lux strain using 10 nM, 100 nM, 1 μM, and 10 μM Ent-testosterone, AIPII, or vehicle.

[0024] [Figure 6] Figure 6 shows the time-dependent quantification of bioluminescence in the MRSAagr-P3lux strain using 10 nM, 100 nM, 1 μM, and 10 μM ent-testosterone or testosterone.

[0025] [Figure 7A-B]Figures 7A–7D show that enantiomerized testosterone inhibited the pathogenicity and agr quorum sensing of Staphylococcus aureus. Figure 7A shows the structures of testosterone and its stereoisomer, enantiomerized testosterone (ent-T). Figures 7B–7C show wild-type Staphylococcus aureus 695 (HG003) treated with 100 nM ent-T, AIP-II, or untreated (vehicle). Figure 7B shows the percentage of Staphylococcus aureus-induced hemolysis, and Figure 7C shows the percentage of Staphylococcus aureus-induced neutrophil killing (C) (n=3). Figure 7D shows qRT-PCR for psmα expression in atopic dermatitis Staphylococcus aureus strains treated with 10 nM testosterone or 10 nM ent-T. Relative psmα expression is normalized to the expression of the housekeeping gene gyrA. The mean ± SEM (error bars) is plotted. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns, not significant by unpaired two-tailed t-test. [Figure 7C] Figures 7A–7D show that enantiomerized testosterone inhibited the pathogenicity and agr quorum sensing of Staphylococcus aureus. Figure 7A shows the structures of testosterone and its stereoisomer, enantiomerized testosterone (ent-T). Figures 7B–7C show wild-type Staphylococcus aureus 695 (HG003) treated with 100 nM ent-T, AIP-II, or untreated (vehicle). Figure 7B shows the percentage of Staphylococcus aureus-induced hemolysis, and Figure 7C shows the percentage of Staphylococcus aureus-induced neutrophil killing (C) (n=3). Figure 7D shows qRT-PCR for psmα expression in atopic dermatitis Staphylococcus aureus strains treated with 10 nM testosterone or 10 nM ent-T. Relative psmα expression is normalized to the expression of the housekeeping gene gyrA. The mean ± SEM (error bars) is plotted. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns, not significant by unpaired two-tailed t-test. [Figure 7D]Figures 7A–7D show that enantiomerized testosterone inhibited the pathogenicity and agr quorum sensing of Staphylococcus aureus. Figure 7A shows the structures of testosterone and its stereoisomer, enantiomerized testosterone (ent-T). Figures 7B–7C show wild-type Staphylococcus aureus 695 (HG003) treated with 100 nM ent-T, AIP-II, or untreated (vehicle). Figure 7B shows the percentage of Staphylococcus aureus-induced hemolysis, and Figure 7C shows the percentage of Staphylococcus aureus-induced neutrophil killing (C) (n=3). Figure 7D shows qRT-PCR for psmα expression in atopic dermatitis Staphylococcus aureus strains treated with 10 nM testosterone or 10 nM ent-T. Relative psmα expression is normalized to the expression of the housekeeping gene gyrA. The mean ± SEM (error bars) is plotted. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns, not significant by unpaired two-tailed t-test.

[0026] [Figure 8A] Figures 8A and 8B show that enantiomerized testosterone (ent-T) inhibits the expression of pathogenic factors and quorum sensing in Staphylococcus aureus. Figure 8A shows qRT-PCR of Staphylococcus aureus strains obtained from patients with atopic dermatitis 1105 and treated with 10 nM or ent-T, a stereoisomer of testosterone (n=3). Expression of the target gene RNAIII normalized to the expression of the housekeeping gene gyrA. Mean ± SEM (error bars) are plotted. *Unpaired two-sided t-test *p<0.05. Figure 8B shows representative bioluminescence images of female wild-type mice percutaneously infected with 1 × 10⁶ CFU agr-P3 Staphylococcus aureus reporter, treated with testosterone, ent-T, vehicle, or untreated (n=4). [Figure 8B]Figures 8A and 8B show that enantiomerized testosterone (ent-T) inhibits the expression of pathogenic factors and quorum sensing in Staphylococcus aureus. Figure 8A shows qRT-PCR of Staphylococcus aureus strains obtained from patients with atopic dermatitis 1105 and treated with 10 nM or ent-T, a stereoisomer of testosterone (n=3). Expression of the target gene RNAIII normalized to the expression of the housekeeping gene gyrA. Mean ± SEM (error bars) are plotted. *Unpaired two-sided t-test *p<0.05. Figure 8B shows representative bioluminescence images of female wild-type mice percutaneously infected with 1 × 10⁶ CFU agr-P3 Staphylococcus aureus reporter, treated with testosterone, ent-T, vehicle, or untreated (n=4).

[0027] [Figure 9A] Figures 9A–9D show that testosterone and DHT activate quorum sensing readout in Staphylococcus aureus, but other hormone classes do not. Figure 9A shows in vitro bioluminescence of agr reporter (HG003 agrP3::lux) after treatment with 10 nM testosterone, DHT, progesterone, or estradiol. Figure 9B shows in vitro bioluminescence of agr reporter (HG003 agrP3::lux) after treatment with DHT. Figure 9C shows in vitro bioluminescence of agr reporter (HG003 agrP3::lux) after treatment with progesterone. Figure 9D shows in vitro bioluminescence of agr reporter (HG003 agrP3::lux) after treatment with AIP-I or estradiol. [Figure 9B-C]Figures 9A–9D show that testosterone and DHT activate quorum sensing readout in Staphylococcus aureus, but other hormone classes do not. Figure 9A shows in vitro bioluminescence of agr reporter (HG003 agrP3::lux) after treatment with 10 nM testosterone, DHT, progesterone, or estradiol. Figure 9B shows in vitro bioluminescence of agr reporter (HG003 agrP3::lux) after treatment with DHT. Figure 9C shows in vitro bioluminescence of agr reporter (HG003 agrP3::lux) after treatment with progesterone. Figure 9D shows in vitro bioluminescence of agr reporter (HG003 agrP3::lux) after treatment with AIP-I or estradiol. [Figure 9D] Figures 9A–9D show that testosterone and DHT activate quorum sensing readout in Staphylococcus aureus, but other hormone classes do not. Figure 9A shows in vitro bioluminescence of agr reporter (HG003 agrP3::lux) after treatment with 10 nM testosterone, DHT, progesterone, or estradiol. Figure 9B shows in vitro bioluminescence of agr reporter (HG003 agrP3::lux) after treatment with DHT. Figure 9C shows in vitro bioluminescence of agr reporter (HG003 agrP3::lux) after treatment with progesterone. Figure 9D shows in vitro bioluminescence of agr reporter (HG003 agrP3::lux) after treatment with AIP-I or estradiol.

[0028] [Figure 10A] Figure 10A shows a series of photographs of mice that were shaved with tape and treated with MRSA, taken on days 0, 1, and 2.

[0029] [Figure 10B]Figure 10B is a series of line graphs showing the effects of in vivo MRSA treatment on bioluminescence (upper panel) and the effect of in vivo MRSA on TEWL (transepidermal water loss), a marker of skin barrier function (lower panel).

[0030] [Figure 11A] Figure 11A is a series of photographs of male mice treated and applied with the AH2659 agrP3 reporter strain, with application of vehicle, 10 nanomoles of ent-testosterone, or 100 nanomoles of ent-testosterone, showing agrP3 brightness at 0, 1, 2, 3, 4, 5, 6, 7, and 8 hours after treatment.

[0031] [Figure 11B] Figure 11B is a line graph showing agrP3 brightness at 0, 1, 2, 3, 4, 5, 6, 7, and 8 hours after treatment with the AH2659 agrP3 reporter strain and treatment with the vehicle, 10 nanomoles of ent-testosterone, or 100 nanomoles of ent-testosterone.

[0032] [Figure 11C] Figure 11C is a bar graph showing the average weight of mice on the day of infection with the AH2659 agrP3 reporter strain in different treatment groups.

[0033] [Figure 11D] Figure 11D is a dot graph showing TEWL in treatment groups with different infection dates using the AH2659 agrP3 reporter strain.

[0034] [Figure 12] Figure 12 is a line graph showing the time course of agrP3 brightness at progressively increasing concentrations of Ent-testosterone, with and without testosterone.

[0035] The drawings do not limit this disclosure to any particular embodiment disclosed and described herein. The drawings are not necessarily to scale, and instead, the emphasis is on illustrating the principles of any particular embodiment of this disclosure. [Modes for carrying out the invention]

[0036] Detailed explanation The following detailed description refers to the accompanying drawings illustrating various aspects of the disclosure. The drawings and descriptions are intended to illustrate aspects of the disclosure in sufficient detail to enable those skilled in the art to practice the disclosure. Other components may be used and modifications may be made without departing from the scope of the disclosure. Therefore, the following description should not be constrained.

[0037] This disclosure is based in part on the identification that enantiomer testosterone (ENT-testosterone) can inhibit bacteria and may be used as a therapeutic agent for treating bacterial infections or conditions complicated by bacterial infections. ENT-testosterone has been found to inhibit the expression of pathogenic factors in Staphylococcus type I, II, III, and IV strains. Furthermore, ENT-testosterone has been found to reduce quorum sensing by reducing the activity of accessory gene regulator C histidine kinase (agrC). Compositions and methods of use of ENT-testosterone for the treatment or prevention of bacterial infections or conditions complicated by bacterial infections are also provided herein.

[0038] I. Terminology For the purpose of facilitating an understanding of the principles of this disclosure, preferred embodiments are referenced herein and specific terminology is used to illustrate them. Unless otherwise defined, all technical and scientific terms used herein have the meanings generally understood by those skilled in the art to which the invention pertains.

[0039] As used herein, the articles “a” and “an” are used to refer to one or more (i.e., at least one) of the grammatical objects of the article. For example, “an element” means at least one element and may include more than one element.

[0040] "Approximately" is used to provide flexibility to the endpoints of a numerical range by indicating that a given value can be "slightly above" or "slightly below" the endpoint without affecting the desired result. In relation to a number, the term "approximately" means that the number can vary by plus or minus 5% or less.

[0041] Throughout this specification, unless the context requires otherwise, the words “comprise” and “include” and their variations (e.g., “comprises,” “comprising,” “includes,” “including”) are understood to include the inclusion of a specified single component, feature, element, or step, or a group of components, features, elements, or steps, but not the exclusion of any other single integer or step, or a group of integers or steps.

[0042] As used herein, “and / or” encompasses every possible combination of one or more of the related enumerated items, as well as the absence of any combination that would be interpreted as an alternative ("or").

[0043] As used herein, the transitional phrase “consisting essentially of” (and its grammatical variant) should be interpreted as encompassing the enumerated materials or steps “that do not substantially affect the basic and novel characterizations of the claimed invention.” Thus, as used herein, the term “consisting essentially of” should not be interpreted as equivalent to “comprising.”

[0044] Furthermore, this disclosure also intends that in some embodiments, any feature or combination of features described herein may be excluded or omitted. For illustrative purposes, where this specification expresses that a certain complex comprises components A, B, and C, it is specifically intended that any one of A, B, or C, or any combination thereof, may be omitted and discarded, individually or in any combination.

[0045] The enumeration of value ranges in this specification is intended merely as a simple way to refer individually to each distinct value within that range unless otherwise specifically indicated herein, and each distinct value is incorporated herein as if it were individually listed herein. For example, if the concentration range is specified as 1% to 50%, then values ​​such as 2% to 40%, 10% to 30%, or 1% to 3% are intended to be explicitly listed herein. These are merely examples of what is specifically intended, and all possible combinations of numerical values ​​between the listed minimum and maximum values, and including them, should be considered to be explicitly indicated herein.

[0046] As used herein, “treatment,” “therapy,” and / or “treatment regimen” refer to clinical interventions made in response to a disease, disorder, or physiological condition indicated by or that a patient may have. The objectives of a treatment include alleviating or preventing symptoms, slowing or halting the progression or worsening of a disease, disorder, or condition, and / or achieving remission of the disease, disorder, or condition.

[0047] As used herein, “prevent” or “prevention” means eliminating or delaying the onset of a particular disease, disorder, or physiological condition, or reducing the degree of severity of a particular disease, disorder, or physiological condition relative to the time and / or degree of onset or severity in the absence of intervention.

[0048] The term "effective dose" or "therapeutic effective dose" refers to a quantity sufficient to produce a beneficial or desirable biological and / or clinical outcome.

[0049] As used herein, “individual,” “subject,” “host,” and “patient” may be used interchangeably herein and refer to any mammalian subject, e.g., human, pet, livestock, horse, or other animal, for which diagnosis, treatment, prevention, or therapy is desired. As used herein, the terms “subject” and “patient” may be used interchangeably herein and refer to both human and non-human animals. The term “non-human animal” in this disclosure includes all vertebrates, e.g., mammals and non-mammals, e.g., non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, etc. In one embodiment, the subject may be a rodent, e.g., mouse, rat, guinea pig, etc. In another embodiment, the subject may be a domesticated animal. Non-limiting examples of suitable domesticated animals may include pigs, cattle, horses, goats, sheep, llamas, and alpacas. In yet another embodiment, the subject may be a companion animal. Non-exclusive examples of companion animals may include pets such as dogs, cats, rabbits, and birds. In yet another embodiment, the subject may be zoo animals. As used herein, “zoo animals” refers to animals that may be found in a zoo. Such animals may include non-human primates, large cats, wolves, and bears. In some embodiments, the subject may be a human. In other embodiments, the subject may be a human requiring treatment for a bacterial infection.

[0050] As used herein, “quorum sensing” refers to bacterial signaling and response. Quorum sensing enables bacterial species to sense their own numbers and regulate gene expression according to population density. Quorum sensing is a cell-density-dependent regulation of genes involving freely diffusible molecules synthesized by cells, called autoinducing substances or autoinducing peptides (AIPs).

[0051] As used herein, “accessory gene regulator” or “agr” refers to an overall regulator of pathogenicity. The agr system, as shown in Figure 1, activates the receptor histidine kinase AgrC in the bacterial cell membrane using a small secreted autoinducible peptide (AIP). AgrC phosphorylates the transcription factor AgrA, which then activates transcription at the P2 and P3 promoters of the operon. Activation of P3 drives the production of RNAIII, an effector of the operon, which regulates the expression of more than 200 pathogenic genes that contribute to invasive infection. Staphylococcus aureus isolates possess one of four agr alleles (type I, type II, type III, or type IV), each encoding a factor that secretes a unique AIP (AIP1, AIP2, AIP3, or AIP4, respectively) detected by the congeneral AgrC histidine kinase. Each Staphylococcus isolate contains only a single copy of the agr system, and each specific species produces different types of AIP signals through variants of the agrBDCA operon. Staphylococcus aureus isolates possess one of four alleles, and isolates possessing any one of these four alleles can cause human disease.

[0052] Where used herein, the interchangeable terms “enantiomer-testosterone,” “Ent-testosterone,” or “Ent-17α-hydroxyandrosta-4-en-3-one” refer to the enantiomer of testosterone, the major male hormone. Enantiomers are pairs of stereoisomers called anticonducting cells, optical anticonducting cells, or optical isomers. They are mirror images of each other and one cannot be superimposed on the other. Enantiomers have the same connectivity but opposite three-dimensional structures. Testosterone is a steroid hormone, primarily produced in males by Leydig cells in the testes and in females in smaller amounts by the ovaries. Small amounts of the hormone are also synthesized in the adrenal cortex of both sexes.

[0053] As used herein, “expression,” “level,” or “level of expression” refers to the amount of a particular protein present in a sample. The amount may be the number, proportion, or percentage of the analyte compared to a control sample. The amount may be absolute or relative.

[0054] As used herein, “Skin and Soft Tissue Infections” or “SSTI” refers to bacterial infections of the skin, muscles, and connective tissues such as ligaments and tendons. Non-exclusive examples of SSTIs include abscesses, cellulitis, folliculitis, MRSA (methicillin-resistant Staphylococcus aureus) infections, and Staph infections (Staphylococcus aureus), erysipelas, impetigo, boils, and carbuncles.

[0055] A biological specimen may be any biological tissue, fluid, or cell from a subject. The specimen may be solid or fluid. The specimen may be a heterogeneous population of cells. Non-limiting examples of suitable biological specimens include sputum, serum, blood, blood cells (e.g., leukocytes), biopsy, urine, ascites, pleural fluid, or cells derived therefrom. Biopsies may be fine-needle aspiration biopsy, core needle biopsy, vacuum-assisted biopsy, open surgical biopsy, thin section biopsy, punch biopsy, incision biopsy, curettage biopsy, or deep shave biopsy. Biological specimens may also include tissue sections, such as frozen sections or formalin-fixed sections, taken for histological purposes. The specimen may be skin tissue, tissue surrounding an infection, or non-infected tissue. Methods for recovering biological specimens from a subject are well known in the art.

[0056] Samples from subjects may be collected one or more times before, during, and / or after diagnosis. In some embodiments, samples may be collected from subjects before, during, and / or after treatment for a bacterial infection. In some embodiments, samples may be collected before, during, and / or after administration of treatment. In addition, samples may be collected repeatedly at multiple stages after the initial sample collection.

[0057] In some embodiments, control samples may be collected from healthy subjects. In some embodiments, control samples may include uninfected cells. In some embodiments, uninfected cells may be from the same histological type as infected cells. For example, if infected cells are from skin, uninfected cells may be from healthy skin tissue. In some embodiments, controls may include mean levels in samples from subjects prior to the onset of infection. In some embodiments, control samples may be samples from subjects prior to diagnosis or treatment. In some embodiments, controls are one or more individuals with similar characteristics to those with infection. In some embodiments, controls may be the average of a combination of different healthy sources (e.g., multiple healthy control subjects). In some embodiments, control samples may be pooled samples.

[0058] As used herein, “effective dose” and “effective amount” may refer to an amount sufficient to achieve a desired outcome, such as the treatment and / or prevention of a disease or disorder (e.g., a bacterial infection). As used herein, “effective dose” and “effective amount” may refer to an amount sufficient to achieve a desired effect against an undesirable condition (e.g., a bacterial infection).

[0059] In some embodiments, “therapeutic dose” means an amount of the disclosed composition that (i) treats a disease or disorder (e.g., a bacterial infection), (ii) reduces, improves or eliminates one or more symptoms associated with a disease or disorder (e.g., a bacterial infection), or (iii) delays the onset of one or more symptoms of a disease or disorder (e.g., a bacterial infection). The specific therapeutic dose level for any particular patient depends on a variety of factors, including the type of bacterial infection being treated; the disclosed composition used; the disclosed method used; the patient’s age, weight, overall health, sex, and diet; the time of administration; the route of administration; the rate of elimination of the disclosed composition used; the duration of treatment; drugs used in combination with or concurrently with the disclosed composition used; and other similar factors well known in the medical field. For example, it is well within the art of the art to start with a dose of the disclosed composition at a level lower than necessary to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. Where desired, the effective daily dose may be divided into multiple doses for the purpose of administration. Therefore, a single dose of a disclosed composition, disclosed pharmaceutical preparation, disclosed therapeutic agent, or combination thereof may contain such an amount or a fraction thereof to constitute a daily dose. Dosage may be adjusted by the individual physician in any contraindications. Dosage may vary and may be administered in doses of one or more doses per day for one or several days. Guidance on appropriate dosages for a given class of medicinal products may be found in the literature. Furthermore, in various embodiments, preparations may be administered in a “preventive effective dose,” that is, an amount effective for the prevention of signs or symptoms associated with a disease or disorder (e.g., a bacterial infection).

[0060] II. Ent-Testosterone This disclosure encompasses ent-testosterone, which can inhibit bacteria. Ent-testosterone inhibits the expression of pathogenic factors in pathogenic microorganisms, such as bacteria of the genus Staphylococcus.

[0061] In some embodiments, the Ent-testosterone disclosed herein has the following chemical structure: [ka]

[0062] In certain embodiments, Ent-testosterone can block, inhibit, or reduce the activity of agrC. In certain embodiments, the Ent-testosterone described herein can reduce agrC activity compared to the agrC activity in the absence of the Ent-testosterone described herein. In certain embodiments, the Ent-testosterone described herein can reduce agrC activity by about 5% to about 99% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%) compared to the agrC activity in the absence of the Ent-testosterone described herein.

[0063] In certain embodiments, Ent-testosterone may block, inhibit, or reduce the expression of one or more pathogenicity-associated protein genes or pathogenic factors in bacteria. In some embodiments, the pathogenicity-associated protein gene is accessory gene regulator (agr) type I. In some embodiments, the pathogenicity-associated protein gene is accessory gene regulator (agr) type II. In some embodiments, the pathogenicity-associated protein gene is accessory gene regulator (agr) type III. In some embodiments, the pathogenicity-associated protein gene is accessory gene regulator (agr) type IV. In certain embodiments, the Ent-testosterone described herein may reduce the expression of one or more pathogenic factors in bacteria compared to the expression of one or more pathogenic factors in bacteria in the absence of the Ent-testosterone described herein. In certain embodiments, the Ent-testosterone described herein may reduce the expression of one or more pathogenic factors in bacteria by about 5% to about 99% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%) compared to the expression of one or more pathogenic factors in bacteria in the absence of the Ent-testosterone described herein. In some embodiments, the Ent-testosterone described herein may reduce the expression of one or more pathogenic factors in bacteria by at least about 10%.

[0064] In certain embodiments, Ent-testosterone can block, inhibit, or reduce bacterial quorum sensing. In certain embodiments, the Ent-testosterone described herein can reduce bacterial quorum sensing compared to bacterial quorum sensing in the absence of the Ent-testosterone described herein. In certain embodiments, the Ent-testosterone described herein can reduce bacterial quorum sensing by about 5% to about 99% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%) compared to bacterial quorum sensing in the absence of the Ent-testosterone described herein. In some embodiments, the Ent-testosterone described herein may reduce bacterial quorum sensing by at least about 10%.

[0065] In some embodiments, ent-testosterone can block, inhibit, or reduce bacterial growth. In certain embodiments, the ent-testosterone described herein can reduce bacterial growth compared to bacterial growth in the absence of the ent-testosterone described herein. In certain embodiments, the ent-testosterone described herein can reduce bacterial growth by about 5% to about 99% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%) compared to bacterial growth in the absence of the ent-testosterone described herein. In some embodiments, the ent-testosterone described herein can reduce bacterial growth by at least about 10%.

[0066] In some embodiments, ent-testosterone can block, inhibit, or reduce bacterial infections. In certain embodiments, the ent-testosterone described herein may reduce bacterial infections compared to bacterial infections in the absence of the ent-testosterone described herein. In certain embodiments, the ent-testosterone described herein may reduce bacterial infections by about 5% to about 99% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%) compared to bacterial infections in the absence of the ent-testosterone described herein. In some embodiments, the ent-testosterone described herein may reduce bacterial infections by at least about 10%.

[0067] In some embodiments, the bacterium may be any bacterium having quorum sensing. In some embodiments, the bacterium may be any bacterium having an agrC signaling pathway. In some embodiments, the bacterium is a bacterium from the genus Staphylococcus. In some embodiments, the bacterium is Staphylococcus aureus or Staphylococcus epidermidis. In some embodiments, Staphylococcus aureus is methicillin-resistant Staphylococcus aureus (MRSA), methicillin-susceptible Staphylococcus aureus (MSSA), vancomycin-intermediate resistant Staphylococcus aureus (VISA), or vancomycin-resistant Staphylococcus aureus (VRSA). In some embodiments, Staphylococcus aureus is Staphylococcus aureus strain LAC, Staphylococcus aureus strain USA 100, Staphylococcus aureus strain MW2, or Staphylococcus aureus strain MN EV.

[0068] III. Composition In some embodiments, compositions comprising ent-testosterone are provided herein. The compositions of this disclosure may be pharmaceutical compositions. In some embodiments, the pharmaceutical compositions of this disclosure may comprise ent-testosterone and at least one pharmaceutically acceptable carrier and / or excipient. Pharmaceutically acceptable carriers or excipients suitable for the compositions described herein are well known to those skilled in the art for use in preserving active ingredients such as hormones and delivering them to any mammalian subject, including humans and other mammals.

[0069] In some embodiments, the compositions described herein may be administered to subjects in need. In some embodiments, the subjects are human. In some embodiments, the subjects have, are suspected of having, or are at risk of having, a bacterial infection or a condition complicated by bacterial colonization. In some embodiments, the subjects have, or are at risk of having, a skin and soft tissue infection (SSTI). In some embodiments, the subjects have, or are at risk of having, atopic dermatitis, folliculitis, pemphigus, or bullous pemphigoid. The compositions disclosed herein can inhibit bacterial growth and / or activity. The bacteria may be any bacteria having an agrC signaling pathway. In some embodiments, the bacteria may be from the genus Staphylococcus. In some embodiments, the bacteria may be Staphylococcus aureus or Staphylococcus epidermidis. In some embodiments, Staphylococcus aureus is methicillin-resistant Staphylococcus aureus (MRSA), methicillin-susceptible Staphylococcus aureus (MSSA), vancomycin-intermediate resistant Staphylococcus aureus (VISA), or vancomycin-resistant Staphylococcus aureus (VRSA). In some embodiments, Staphylococcus aureus is Staphylococcus aureus strain LAC, Staphylococcus aureus strain USA 100, Staphylococcus aureus strain MW2, or Staphylococcus aureus strain MN EV.

[0070] Therefore, in some embodiments, ent-testosterone can be formulated into pharmaceutical compositions. The compositions can be formulated in various forms adapted to preferred routes of administration. Thus, the compositions can be administered via known routes, including, for example, oral, parenteral (e.g., intradermal, transdermal, subcutaneous, intramuscular, intravenous, intraperitoneal, etc.), or topical (e.g., intranasal, intrapulmonary, intramammary, intravaginal, intrauterine, intradermal, transrectal, etc.). The compositions can be administered to mucosal surfaces, for example, by administration to the nasal or respiratory mucosa (e.g., by spray or aerosol). The compositions can also be administered via sustained or delayed release and / or eluted in combination with or from medical bandages, such as bandages.

[0071] In certain embodiments, pharmaceutical compositions may be provided in any preferred form, including but not limited to liquids, suspensions, emulsions, sprays, aerosols, or mixtures of any form. For example, formulations may be delivered in conventional topical dosage forms such as creams, ointments, aerosol formulations, non-aerosol sprays, gels, and lotions. The compositions may further contain one or more additives, such as adjuvants, skin penetration enhancers, colorants, fragrances, flavorings, moisturizers, and thickeners.

[0072] In certain embodiments, the composition used in this method may, in the form of a lyophilized formulation or aqueous solution, comprise pharmaceutically acceptable carriers, excipients, and / or stabilizers. In some embodiments, the acceptable carriers, excipients, and / or stabilizers are non-toxic to the recipient at the dosage and concentration used and may include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens, e.g., methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); and low molecular weight (less than about 10 residues) polypeptides. ;Proteins such as serum albumin, gelatin, or immunoglobulins;Hydrophilic polymers such as polyvinylpyrrolidone;Amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine;Monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextran;Chelating agents such as EDTA;Sugars such as sucrose, mannitol, trehalose, or sorbitol;Salt-forming counterions such as sodium;Metal complexes (e.g., Zn-protein complexes);And / or nonionic surfactants, such as TWEEN®, PLURONICS, or polyethylene glycol (PEG).

[0073] In some embodiments, the compositions described herein may have liposomes containing ENT-testosterone. In some embodiments, liposomes for use herein may be produced by reverse-phase evaporation using a lipid composition having phosphatidylcholine, cholesterol, and PEG-derivativeized phosphatidylethanolamine (PEG-PE). In some embodiments, liposomes for use herein may be extruded through a filter of a specified pore size to obtain liposomes having a desired diameter.

[0074] In some embodiments, ent-testosterone may be encapsulated in microcapsules prepared, for example, by coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions.

[0075] In some embodiments, the compositions herein may be sterile. This can be easily achieved, for example, by filtration through a sterile filtration membrane. In some embodiments, the compositions herein may be placed in a container having a sterile access port, such as an intravenous solution bag or vial having a stopper that can be punctured by a subcutaneous needle.

[0076] In certain embodiments, the compositions described herein may be in unit dosage forms such as tablets, pills, capsules, powders, granules, liquids or suspensions, or suppositories for oral, parenteral, or rectal administration, or administration by inhalation or inhalation. Formulations may be conveniently presented in unit dosage forms and may be prepared by methods well known in the field of pharmaceuticals. A method for preparing a composition comprising a pharmaceutically acceptable carrier comprises the step of associating ENT-testosterone with a carrier constituting one or more accessory components. Generally, formulations may be prepared by uniformly and / or tightly associating an active compound with a liquid carrier, a micronized solid carrier, or both, and then, if necessary, shaping the product into a desired formulation.

[0077] In some embodiments, the emulsion compositions of this specification may be prepared by mixing Ent-testosterone with Intralipid® or its components (soybean oil, egg phospholipid, glycerol, and water).

[0078] Suitable emulsions can be prepared using commercially available lipid emulsions such as Intralipid®, Liposyn®, Infonutrol®, Lipofundin®, and / or Lipiphysan®. The active ingredient may be dissolved in a pre-mixed emulsion composition, or in an emulsion formed by mixing oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil, or almond oil) and phospholipids (e.g., egg phospholipid, soybean phospholipid, or soybean lecithin) with water. It will be understood that other components, such as glycerol or glucose, may be added to adjust the tonicity of the emulsion. Suitable emulsions typically contain up to about 20% oil, for example, about 5% to about 20%. The lipid emulsion can consist of lipid droplets approximately 0.1 μm to 1.0 μm in size, particularly approximately 0.1 μm to 0.5 μm, and has a pH in the range of approximately 5.5 to 8.0.

[0079] In some embodiments, the compositions herein for inhalation or intranasal administration may comprise solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents, or mixtures thereof, as well as powders. In some embodiments, the liquid or solid compositions herein may comprise the above-mentioned preferred pharmaceutically acceptable excipients. In other embodiments, the compositions may be administered orally or via nasal respiratory routes for topical or systemic effects.

[0080] In some embodiments, the composition may be in a sterile, pharmaceutically acceptable solvent and may be atomized by the use of a gas. In some embodiments, the atomized solutions of this specification may be inhaled directly from an atomizing device, or the atomizing device may be attached to a face mask, tent, or intermittent positive pressure respirator. In some embodiments, the solutions, suspensions, or powder compositions of this specification may be administered preferably orally or nasally from a device that delivers the formulation in an appropriate manner.

[0081] In certain embodiments, the compositions disclosed herein may include a buffer system. As used herein, “buffer system” is a composition comprising one or more buffers, and “buffer” is a compound used to resist changes in pH when an acid or alkali is diluted or added. Buffers include, but are not limited to, potassium metaphosphate, potassium phosphate, monobasic sodium acetate, and sodium citrate anhydrous and dihydrate, as well as other materials known to those skilled in the art. In some embodiments, any pharmaceutically acceptable organic or inorganic buffer may be used. In some embodiments, the pharmaceutical compositions disclosed herein may contain one or more buffers in a total amount of at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% of the total weight of the composition. In some embodiments, the pharmaceutical compositions disclosed herein may contain one or more buffering agents in an amount of about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% of the total weight of the composition.

[0082] In some embodiments, the amount of one or more buffering agents may depend on the desired pH level of the composition. In some embodiments, the pharmaceutical compositions disclosed herein may have a pH of about 6 to about 9. In some embodiments, the pharmaceutical compositions disclosed herein may have a pH greater than about 8, greater than about 7.5, greater than about 7, greater than about 6.5, or greater than about 6.

[0083] In certain embodiments, the compositions disclosed herein may contain one or more preservatives. As used herein, “preservative” means an agent or combination of agents that inhibits, reduces, or eliminates bacterial growth in a pharmaceutical dosage form. Non-limiting examples of preservatives include nipagin, nipazole, isopropyl alcohol, and combinations thereof. In some embodiments, any pharmaceutically acceptable preservative may be used. In some embodiments, the pharmaceutical compositions disclosed herein may contain one or more preservatives in a total amount of at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% of the total weight of the composition. In some embodiments, the pharmaceutical compositions disclosed herein may contain one or more preservatives in a total amount of about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% of the total weight of the composition.

[0084] In certain embodiments, the compositions disclosed herein may comprise one or more surface-acting reagents or detergents. In some embodiments, the surface-acting reagents or detergents may be synthetic, natural, or semi-synthetic. In some embodiments, the compositions disclosed herein may comprise anionic detergents, cationic detergents, zwitterionic detergents, ampholytic detergents, amphoteric detergents, nonionic detergents having a steroid skeleton, or combinations thereof. In some embodiments, the compositions disclosed herein may comprise one or more surface-acting reagents or detergents in a total amount of at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% of the total weight of the composition. In some embodiments, the compositions disclosed herein may contain one or more of a surface-acting reagent or cleaning agent in a total amount of about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% of the total weight of the composition.

[0085] In certain embodiments, the compositions disclosed herein may contain one or more stabilizers. As used herein, “stabilizer” means a compound used to stabilize an active agent against physical, chemical, or biochemical processes that could otherwise reduce the therapeutic activity of the agent. Suitable stabilizers include, but are not limited to, succinic anhydride, albumin, sialic acid, creatinine, glycine and other amino acids, niacinamide, sodium acetyltryptophanate, zinc oxide, sucrose, glucose, lactose, sorbitol, mannitol, glycerol, polyethylene glycol, sodium caprylate, and sodium saccharin, as examples. In some embodiments, the compositions disclosed herein may contain one or more stabilizers in a total amount of at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% of the total weight of the composition. In some embodiments, the compositions disclosed herein may contain one or more stabilizers in a total amount of about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% based on the total weight of the composition.

[0086] In some embodiments, the compositions disclosed herein may contain one or more isotonic agents. As used herein, “isotonic agent” refers to a compound that can be used to adjust the tonicity of a liquid formulation. Suitable isotonic agents include, but are not limited to, glycerin, lactose, mannitol, dextrose, sodium chloride, sodium sulfate, sorbitol, and trehalose, which are known to those skilled in the art. The volumetric osmolality of a composition may be expressed in milliosmolality / liter (mOsm / L). The volumetric osmolality may be measured using methods commonly known in the art. In some embodiments, the vapor pressure lowering method is used to calculate the volumetric osmolality of the compositions disclosed herein. In some embodiments, the amount of one or more isotonic agents constituting the pharmaceutical compositions disclosed herein may result in composition volume osmolality concentrations of about 150 mOsm / L to about 500 mOsm / L, about 250 mOsm / L to about 500 mOsm / L, about 250 mOsm / L to about 350 mOsm / L, about 280 mOsm / L to about 370 mOsm / L, or about 250 mOsm / L to about 320 mOsm / L. In some embodiments, the compositions according to this specification may have a gravimetric osmolality in the range of about 100 mOsm / kg to about 1000 mOsm / kg, about 200 mOsm / kg to about 800 mOsm / kg, about 250 mOsm / kg to about 500 mOsm / kg, or about 250 mOsm / kg to about 320 mOsm / kg, or about 250 mOsm / kg to about 350 mOsm / kg, or about 280 mOsm / kg to about 320 mOsm / kg. In some embodiments, the pharmaceutical compositions described herein may have volumetric osmolality concentrations of about 100 mOsm / L to about 1000 mOsm / L, about 200 mOsm / L to about 800 mOsm / L, about 250 mOsm / L to about 500 mOsm / L, about 250 mOsm / L to about 350 mOsm / L, about 250 mOsm / L to about 320 mOsm / L, or about 280 mOsm / L to about 320 mOsm / L.In some embodiments, the pharmaceutical compositions disclosed herein may contain one or more tonicity modifiers in a total amount of at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% of the total weight of the composition. In some embodiments, the pharmaceutical compositions disclosed herein may contain one or more tonicity modifiers in a total amount of about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% of the total weight of the composition.

[0087] In some embodiments, the concentrations of Ent-testosterone disclosed herein may be predetermined or standard concentrations. In some embodiments, the Ent-testosterone disclosed herein may be at concentrations of approximately 1 microgram / milliliter (mg / ml) to approximately 500 mg / ml, approximately 1 mg / ml to approximately 250 mg / ml, approximately 1 mg / ml to approximately 200 mg / ml, approximately 1 mg / ml to approximately 150 mg / ml, approximately 1 mg / ml to approximately 100 mg / ml, approximately 1 mg / ml to approximately 75 mg / ml, approximately 1 mg / ml to approximately 50 mg / ml, approximately 0.1 mg / ml to approximately 100 mg / ml, or other preferred concentrations. In some embodiments, the Ent-testosterone is lyophilized. In some embodiments, the Ent-testosterone is diluted to a preferred concentration in a preferred solution before administration.

[0088] In certain embodiments, the composition may be provided in any preferred form, including but not limited to liquids, suspensions, emulsions, sprays, aerosols, or mixtures of any form. In some embodiments, Ent-testosterone is formulated for topical application. For example, the formulation may be delivered as a cream, ointment, aerosol, non-aerosol spray, gel, lotion, etc. The composition may further contain one or more additives, including, for example, adjuvants, skin penetration enhancers, colorants, fragrances, flavorings, moisturizers, and thickeners. In some embodiments, the compositions disclosed herein contain Ent-testosterone and petrolatum.

[0089] In certain embodiments, the compositions for this disclosure may comprise a patch applied to an area of ​​skin to be treated. As used herein, “patch” comprises at least a topical composition comprising ent-testosterone and a covering layer, thereby the patch may be placed over an area of ​​skin to be treated. The patch may be designed to maximize drug delivery through the stratum corneum to the epidermis or dermis, reduce delay time, promote uniform absorption, and / or reduce mechanical friction. In certain embodiments, where the intended use involves the treatment of a skin infection, the patch is designed to minimize absorption into the circulatory system. Preferably, the patch components are similar in viscoelastic properties to those of the skin and conform to the skin during movement to prevent excessive shearing and delamination. The advantages of patches, including topical formulations, over conventional methods of administration include (i) dose control by the patch's surface area, (ii) constant administration rate, (iii) longer duration of action (ability to adhere to the skin for 1, 3, 7 days or more), (iv) improved patient compliance, (v) non-invasive administration, and (vi) reversible action (i.e., the patch can be easily removed).

[0090] In one embodiment, a patch suitable for use in this disclosure comprises at least: (1) a backing layer and (2) a carrier formulated with the compound of the present invention. Examples of patch systems suitable for practicing the present invention include, but are not limited to, matrix patches, reservoir patches, multi-laminate drug adhesive patches, and monolithic drug adhesive patches. A matrix patch includes, but is not limited to, a matrix containing the composition disclosed herein, an adhesive backing film overlay, and preferably a release liner. In some cases, it may be necessary to include an impermeable layer to minimize drug transfer to the backing film. In some embodiments, the matrix containing the compound of the present invention is retained to the skin by an adhesive overlay. Examples of suitable matrix materials include, but are not limited to, lipophilic polymers such as polyvinyl chloride and polydimethylsiloxane, and hydrophilic polymers such as polyvinylpyrrolidone, polyvinyl alcohol, gelatin-based hydrogels, or polyvinylpyrrolidone / polyethylene oxide mixtures. Suitable release liners include, but are not limited to, occluding, opaque, or transparent polyester films having a thin coating of a pressure-sensitive release liner (e.g., silicone-fluorosilicone and perfluorocarbon polymers).

[0088] In some embodiments, the amount of ent-testosterone administered may vary depending on a variety of factors, including but not limited to the specific microorganism requiring treatment, the subject's weight, physical condition, and / or age, and / or route of administration. Thus, the absolute weight of ent-testosterone contained in a given unit dosage form can vary considerably and depends on factors such as the ent-testosterone used, the species of infectious microorganism, the subject's age, weight, and physical condition, and / or method of administration. Those skilled in the art can easily determine an appropriate amount by considering such factors.

[0089] In some embodiments, the compositions disclosed herein may be administered to provide a target dose of sufficient ent-testosterone, for example, about 100 ng / kg to about 50 mg / kg. In some embodiments, when administered, the compositions provide a target dose of ent-testosterone, for example, about 100 μg / kg to about 5 mg / kg, or about 100 μg / kg to about 1 mg / kg.

[0090] In some embodiments, the compositions described herein may further comprise one or more active or therapeutic agents. Alternatively, ENT-testosterone may be administered to patients in need in combination with the administration of one or more other therapeutic agents. Non-limiting examples of additional active or therapeutic agents include, but are not limited to, antibiotics, anti-inflammatory agents, analgesics, immunomodulators, and anticancer agents. Antibiotics may be bactericidal antibiotics, bacteriostatic antibiotics, and / or combinations of two or more antibiotics. Exemplary antibiotics include, for example, lincosamides (e.g., lincomycin or clindamycin), penicillins (e.g., nafcillin), cephalosporins (e.g., cephthaloline, ceftazidime (alone or in combination with avibactam), or ceftolozane (alone or in combination with tazobactam)), glycopeptides (e.g., vancomycin, oritabancin, dalbavancin), lipopeptides (e.g., daptomycin), aminoglycosides (e.g., gentamicin), oxazolidinones (e.g., linezolid, tedizolid, posizolid, or cycloserine), or tetracyclines (e.g., doxycycline). Exemplary antimicrobial agents include, for example, immunotherapeutic agents, such as antimicrobial antibody treatment and / or antimicrobial cytokine treatment.

[0091] In some embodiments, Ent-testosterone may be administered to a subject before or after the subject exhibits symptoms or clinical signs of a bacterial infection. “Symptoms” refers to any subjective evidence of a disease or patient condition. “Signs” or “clinical signs” refers to objective physical findings relating to a particular condition that may be observed by someone other than the patient. Treatment initiated before the subject exhibits symptoms or clinical signs of an infection may be considered a preventative measure for a subject “at risk” of bacterial infection. As used herein, the term “at risk” refers to a subject that may or may not actually possess the described risk. Thus, for example, a subject “at risk” of an infectious condition is a subject located in an area where there may be exposure to an infectious agent, even if other individuals have been identified as having the infectious condition and / or the subject has not yet exhibited detectable signs of a bacterial infection, and regardless of whether the subject may possess a subclinical amount of bacteria.

[0092] As a result, administration of the composition may be performed before, during, or after the subject first exhibits symptoms or clinical signs of the condition, or before, during, or after the subject first comes into contact with bacteria. Treatment initiated before the subject first exhibits symptoms or clinical signs associated with the condition may reduce the likelihood of the subject experiencing clinical evidence of the condition, reduce the severity of symptoms and / or clinical signs of the condition, and / or completely resolve the condition, compared to a subject who has not been administered the composition. Treatment initiated after the subject first exhibits symptoms or clinical signs associated with the condition may be considered a therapeutic treatment for the subject and may reduce the severity of symptoms and / or clinical signs of the condition, and / or completely resolve the condition, compared to a subject who has not been administered the composition.

[0093] In some embodiments, administration of a composition containing ent-testosterone can block, inhibit, or reduce tissue damage in a subject. In some embodiments, the tissue damage may be damage to the skin or other soft tissue. In some embodiments, administration of a composition containing ent-testosterone can reduce tissue damage in a subject by at least 10%, e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more, compared to tissue damage in a subject before administration of the composition containing ent-testosterone (baseline) or in an untreated control subject. In some embodiments, administration of the disclosed composition containing ent-testosterone reduces tissue damage in a subject by at least about 20%. Tissue damage can be determined using any method known in the art for measuring cell apoptosis or cell viability, including bioluminescence imaging or flow cytometry.

[0094] IV. Treatment Methods This disclosure further provides methods for treating subjects having a bacterial infection or a condition complicated by bacterial colonization.

[0095] In some embodiments, the method includes treating a subject having or at risk of having a bacterial infection, comprising administering a composition comprising ent-testosterone to the subject. The bacterial infection may be caused by bacteria from the genus Staphylococcus. In some embodiments, the bacteria are Staphylococcus aureus or Staphylococcus epidermidis. In some embodiments, Staphylococcus aureus is methicillin-resistant Staphylococcus aureus (MRSA), methicillin-susceptible Staphylococcus aureus (MSSA), vancomycin-intermediate resistant Staphylococcus aureus (VISA), or vancomycin-resistant Staphylococcus aureus (VRSA). In some embodiments, Staphylococcus aureus is Staphylococcus aureus strain LAC, Staphylococcus aureus strain USA 100, Staphylococcus aureus strain MW2, or Staphylococcus aureus strain MN EV.

[0096] In further embodiments, the disclosure encompasses methods for treating subjects having or at risk of having bacterial skin and soft tissue infections (SSTIs), the methods comprising administering a composition comprising ent-testosterone to the subjects. In some embodiments, the SSTIs are abscesses, cellulitis, folliculitis, MRSA (methicillin-resistant Staphylococcus aureus) infections, and Staph infections (Staphylococcus aureus or Staphylococcus epidermidis), erysipelas, impetigo, boils, or carbuncles. The bacteria may be from the genus Staphylococcus. In some embodiments, the bacteria are Staphylococcus aureus. In some embodiments, Staphylococcus aureus is methicillin-resistant Staphylococcus aureus (MRSA), methicillin-susceptible Staphylococcus aureus (MSSA), vancomycin-intermediate resistant Staphylococcus aureus (VISA), or vancomycin-resistant Staphylococcus aureus (VRSA). In some embodiments, Staphylococcus aureus is Staphylococcus aureus strain LAC, Staphylococcus aureus strain USA 100, Staphylococcus aureus strain MW2, or Staphylococcus aureus strain MN EV.

[0097] In another embodiment, the method comprises treating a subject having, or at risk of having, a condition complicated by bacterial colonization, by administering to the subject a composition comprising ent-testosterone. In some embodiments, the condition is atopic dermatitis, folliculitis, pemphigus, bullous pemphigoid, or cutaneous T-cell lymphoma. The bacteria may be from the genus Staphylococcus. In some embodiments, the bacteria are Staphylococcus aureus or Staphylococcus epidermidis. In some embodiments, Staphylococcus aureus is methicillin-resistant Staphylococcus aureus (MRSA), methicillin-sensitive Staphylococcus aureus (MSSA), vancomycin-intermediate resistant Staphylococcus aureus (VISA), or vancomycin-resistant Staphylococcus aureus (VRSA). In some embodiments, Staphylococcus aureus is Staphylococcus aureus strain LAC, Staphylococcus aureus strain USA 100, Staphylococcus aureus strain MW2, or Staphylococcus aureus strain MN EV.

[0098] In some embodiments, the methods disclosed herein include treating subjects who have or are at risk of having a Staphylococcus aureus infection. The methods include administering a composition comprising ent-testosterone to the subjects. Staphylococcus aureus may be methicillin-resistant Staphylococcus aureus (MRSA), methicillin-susceptible Staphylococcus aureus (MSSA), vancomycin-intermediate resistant Staphylococcus aureus (VISA), or vancomycin-resistant Staphylococcus aureus (VRSA). In some embodiments, Staphylococcus aureus strain LAC, Staphylococcus aureus strain USA 100, Staphylococcus aureus strain MW2, or Staphylococcus aureus strain MN EV.

[0099] In another embodiment, the methods disclosed herein include treating a subject having or at risk of having a Staphylococcus epidermidis infection. The methods include administering a composition comprising ent-testosterone to the subject.

[0100] In another embodiment, the method includes limiting damage to the target tissue caused by bacterial pathogenicity-associated proteins by administering a composition containing ent-testosterone to the target. In such embodiments, the tissue is skin tissue or other soft tissue. The bacteria may be from the genus Staphylococcus. In some embodiments, the bacteria are Staphylococcus aureus or Staphylococcus epidermidis. In some embodiments, Staphylococcus aureus is methicillin-resistant Staphylococcus aureus (MRSA), methicillin-sensitive Staphylococcus aureus (MSSA), vancomycin-intermediate resistant Staphylococcus aureus (VISA), or vancomycin-resistant Staphylococcus aureus (VRSA). In some embodiments, Staphylococcus aureus is Staphylococcus aureus strain LAC, Staphylococcus aureus strain USA 100, Staphylococcus aureus strain MW2, or Staphylococcus aureus strain MN EV.

[0101] A method for reducing the pathogenicity of a staphylococcus species is further provided, comprising contacting the staphylococcus species with a composition containing ent-testosterone. In some embodiments, the staphylococcus species is Staphylococcus aureus or Staphylococcus epidermidis. In some embodiments, Staphylococcus aureus is methicillin-resistant Staphylococcus aureus (MRSA), methicillin-sensitive Staphylococcus aureus (MSSA), vancomycin-intermediate resistant Staphylococcus aureus (VISA), or vancomycin-resistant Staphylococcus aureus (VRSA). In some embodiments, Staphylococcus aureus is Staphylococcus aureus strain LAC, Staphylococcus aureus strain USA 100, Staphylococcus aureus strain MW2, or Staphylococcus aureus strain MN EV.

[0102] In some aspects of the method, the disclosed bacteria may be any bacteria having quorum sensing. Any bacteria having an agrC signaling pathway, such as Pseudomonas aeruginosa, are intended to be treated by the methods disclosed herein.

[0103] The subjects may be those at risk of or diagnosed with a bacterial infection, such as a staphylococcal infection. Staphylococcal infections, particularly Staphylococcus aureus infections, can affect various parts of the body and may include skin infections as well as conditions such as osteomyelitis, endocarditis, septic arthritis, pneumonia, abscess formation, gastroenteritis, burn-like skin syndrome, and toxic shock syndrome. Staphylococcus aureus strains are considered a major cause of hospital-acquired infections in the United States. Staphylococcal infections, particularly Staphylococcus aureus infections, caused by strains resistant to commonly used antibiotics are especially serious and life-threatening. Subjects at high risk of staphylococcal infection include, but are not limited to, those with conditions such as diabetes, cancer, vascular disease, eczema, or lung disease, those with burns, or those who have had close contact with someone who has a staphylococcal infection. In a further embodiment, subjects at high risk of staphylococcal infection may include those in the intensive care unit, those who have undergone surgery, those undergoing dialysis, those using catheters, breathing tubes, or feeding tubes, those with medical devices implanted in their bodies such as pacemakers, artificial joints, or heart valves, those with weakened immune systems such as those diagnosed with HIV, those taking therapeutic drugs to prevent organ or tissue transplant rejection, or those undergoing chemotherapy. Subjects may be children or infants attending daycare, kindergarten, or school. In a further embodiment, subjects may be at risk of or have been diagnosed with Staphylococcus epidermidis. Staphylococcus epidermidis infection can cause wound infections, swelling, sinus infections, endocarditis, and other inflammations.

[0104] The methods disclosed herein may include a composition containing an effective amount of ent-testosterone. The effective amount may be a therapeutically effective amount. The composition may be administered as a dose to a subject. In some embodiments of the methods, preferred non-limiting examples of doses according to this disclosure may be about 1 ng / kg to about 5000 mg / kg. However, generally, doses used for the treatment of adults may typically range from about 0.0001 mg / kg / day to about 0.0010 mg / kg / day, about 0.0010 mg / kg / day to about 0.010 mg / kg / day, about 0.010 mg / kg / day to about 0.10 mg / kg / day, about 0.10 mg / kg / day to about 1 mg / kg / day, about 1 mg / kg / day to about 200 mg / kg / day, and about 200 mg / kg / day to about 5000 mg / kg / day. For example, the dosage could range from approximately 1 mg / kg / day to approximately 100 mg / kg / day, such as approximately 2 to 10 mg / kg / day, approximately 10 to 50 mg / kg / day, or approximately 50 to 100 mg / kg / day. The dosage is approximately 1 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, 125 mg / kg, 150 mg / kg, 175 mg / kg, 200 mg / kg, 250 mg / kg, 300 mg / kg, 400 mg / kg, 500 mg / kg, 600 mg / kg, 700 mg / kg, 800 mg / kg, 900 mg / kg, and 1 You can choose from approximately 000 mg / kg, approximately 1100 mg / kg, approximately 1200 mg / kg, approximately 1300 mg / kg, approximately 1400 mg / kg, approximately 1500 mg / kg, approximately 1600 mg / kg, approximately 1700 mg / kg, approximately 1800 mg / kg, approximately 1900 mg / kg, approximately 2000 mg / kg, approximately 2100 mg / kg, approximately 2200 mg / kg, approximately 2300 mg / kg, approximately 2400 mg / kg, approximately 2500 mg / kg, approximately 2600 mg / kg, approximately 2700 mg / kg, approximately 2800 mg / kg, approximately 2900 mg / kg, approximately 3000 mg / kg, approximately 3500 mg / kg, approximately 4000 mg / kg, or approximately 5000 mg / kg.

[0105] The Ent-testosterone disclosed herein may be administered to a subject daily or more than once daily. In some embodiments, the disclosed Ent-testosterone may be administered every 2, 3, 4, 5, 6, 7, 14 days, or every 30 days. The disclosed Ent-testosterone may be administered over periods ranging from about 1 day to about 1 year, about 1 day to about 1 week, about 3 days to about 1 month, about 2 weeks to about 6 months, or about 2 months to about 4 months. The disclosed Ent-testosterone may also be administered over periods of about 1 day, about 7 days, about 30 days, about 60 days, about 120 days, or about 180 days or longer. In some embodiments, the disclosed Ent-testosterone may be administered over periods of about 57 weeks, about 148 weeks, about 208 weeks, or indefinitely.

[0106] Dosage and intervals may be individually adjusted to provide an amount sufficient to maintain a therapeutic or preventive effect. For example, Ent-testosterone may be administered once a week, several times a week (e.g., every other day), once a day, or multiple times a day, depending, among other things, on the mode of administration, the specific indication being treated, and the prescribing physician's discretion. In the case of selective uptake, such as topical administration, the effective local concentration of Ent-testosterone may not be related to plasma concentration. Those skilled in the art can optimize the effective dose without excessive experimentation.

[0107] In some embodiments, the method of administering ent-testosterone results in a reduction of tissue damage in subjects. In some embodiments, the tissue damage may be damage to the skin or other soft tissues. In some embodiments, the method of administering ent-testosterone can reduce tissue damage in subjects by at least 10%, e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more, compared to tissue damage in subjects before ent-testosterone administration (baseline) or in untreated control subjects. In some embodiments, the method of administering ent-testosterone reduces tissue damage in subjects by at least about 20%. Tissue damage can be determined using any method known in the art for measuring cell apoptosis or cell viability, including bioluminescence imaging or flow cytometry.

[0108] In one embodiment, the administration of ent-testosterone results in a reduction of staphylococcal-induced hemolysis in subjects. In several embodiments, the administration of ent-testosterone can reduce staphylococcal-induced hemolysis in subjects by at least 10%, e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more, compared to staphylococcal-induced hemolysis in subjects before ent-testosterone administration (baseline) or in untreated control subjects. In several embodiments, the administration of ent-testosterone reduces staphylococcal-induced hemolysis in subjects by at least about 5%.

[0109] In another embodiment, the administration of ent-testosterone results in a reduction of staphylococcal-induced neutrophil killing in subjects. In some embodiments, the administration of ent-testosterone can reduce staphylococcal-induced neutrophil killing in subjects by at least 10%, e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more, compared to staphylococcal-induced neutrophil killing in subjects before ent-testosterone administration (baseline) or in untreated control subjects. In some embodiments, the administration of ent-testosterone reduces staphylococcal-induced neutrophil killing in subjects by at least about 5%.

[0110] In further embodiments, the disclosed method of administering ent-testosterone results in a reduction of one or more adverse effects caused by bacterial infection, e.g., Staphylococcus aureus in the subject, or a reduction in disease status in the subject. Non-limited examples of adverse effects include hemolysis, toxic shock, biofilm formation, and pathogenicity. In some embodiments, the method of administering ent-testosterone can reduce one or more hemolysis, toxic shock, biofilm formation, and pathogenicity in a subject by at least 10%, e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more, compared to hemolysis, toxic shock, biofilm formation, and / or pathogenicity in a subject before administration of ent-testosterone (baseline) or in an untreated control subject.

[0111] The methods of this disclosure may be administered concurrently with, before, or after, one or more other desired therapeutic agents or medical procedures. The specific combination of therapies (therapeutic agents or procedures) used in a combination regimen will take into account the suitability of the desired therapeutic agents and / or procedures as well as the desired therapeutic effect to be achieved. It will also be understood that the therapies used may achieve a desired effect for the same disorder (e.g., ENT-testosterone may be administered concurrently with another anti-inflammatory agent, immunomodulator, antibiotic, analgesic, or anticancer agent) or a different effect (e.g., control of any adverse effects).

[0112] In some embodiments, the disclosed method may further include modifying one or more doses of Ent-testosterone as described herein. For example, modifying one or more steps of the administration method may include modifying or changing one or more features or aspects of one or more steps of the disclosed method. For example, in some embodiments, the method may be altered by changing the amount of Ent-testosterone administered to a subject, or by changing the frequency of administration of Ent-testosterone to a subject, or by changing the duration of one or more disclosed Ent-testosterone administered to a subject.

[0113] V. Kit This disclosure provides kits containing ent-testosterone for use with the methods of this disclosure. The kits may include a composition containing ent-testosterone and instructions for administering the ent-testosterone to a subject requiring it. The kits may further include other therapeutic agents, such as antibiotics, that can be administered in combination with ent-testosterone. In some embodiments, the kits may further include a sterile, pharmaceutically acceptable carrier, buffer, or other diluent. The kits provided herein generally include instructions for performing the methods. Instructions included in the kit may be attached to the packaging material or included as accompanying documentation. Instructions are typically in writing or printed form, but are not limited thereto. Any medium capable of storing such instructions and communicating them to end users is contemplated by this disclosure. Such mediums include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical media (e.g., CD-ROMs), etc. As used herein, the term “instructions” may include the address of an internet site providing the instructions.

[0114] The disclosed kit may or may not contain additional components and may have a single container containing the disclosed Ent-testosterone, or it may have separate containers for each desired drug. If a combined therapeutic agent is provided, the single solution may be pre-mixed in molar equivalent combinations or with one component in excess of the other. Alternatively, the Ent-testosterone and other therapeutic agent components of the kit may be maintained separately in separate containers before administration to the patient.

[0115] If the components of the kit are provided as one or more liquid solutions, the liquid solutions are preferably aqueous solutions, and sterile aqueous solutions are particularly preferred. However, the components of the kit may also be provided as dry powder(s). If the reagents or components are provided as dry powder, the powder can be reconstituted by the addition of a suitable solvent. The solvent may also be provided in a separate container.

[0116] The kit container generally comprises at least one vial, test tube, flask, bottle, syringe, or other container means in which the disclosed Ent-testosterone and any other desired drugs can be placed, preferably preferably divided equally. If separate components are included, the kit also generally comprises a second vial or other container in which these are placed, enabling the administration of separately designed doses. The kit may also comprise a second / third container means for containing a sterile, pharmaceutically acceptable buffer or another diluent.

[0117] The kit may also include means for administering the disclosed Ent-testosterone to an animal or patient, such as one or more needles or syringes, or eyedroppers, pipettes, or other similar apparatus from which the formulation can be injected into the animal or applied to a diseased area of ​​the body. The kit of the present disclosure also typically includes means for containing vials or of this kind and other components in a sealed state for commercial sale, such as injection-molded or blow-molded plastic containers in which the desired vials and other apparatus are placed and held.

[0118] While several embodiments have been described, it will be recognized by those skilled in the art that various modifications, alternative configurations, and equivalents can be used without departing from the spirit of this disclosure. In addition, some well-known processes and elements have been omitted to avoid unnecessarily obscuring this disclosure. Therefore, this description should not be construed as limiting the scope of this disclosure.

[0119] Those skilled in the art will understand that the embodiments currently disclosed are taught as examples, not as limitations. Therefore, matters included herein or shown in the accompanying drawings should be construed as illustrative and not as limiting. The following claims are intended to encompass all general and specific features described herein, as well as all descriptions of the scope of methods and assemblies, which may be said to be between them as a matter of language. [Examples]

[0120] The following embodiments are included to illustrate aspects of the present disclosure. Those skilled in the art should understand that the techniques disclosed in the following embodiments represent techniques that the inventors have found to function well in the practice of the present disclosure and may thus constitute a preferred form for that practice. However, those skilled in the art should understand that, in light of the present disclosure, many modifications may be made in the specific embodiments disclosed without departing from the spirit and scope of the present disclosure, and still similar or comparable results may be obtained.

[0121] material and method mouse Six-to-nine-week-old C57BL6 / J male and female mice, reared using conventional methods, were purchased from Jackson Laboratory. C57BL6 / J wild-type, K14Cre+ / -(60), and Hsd3b6fl / fl mice were bred and maintained in a specific pathogen-free (SPF) barrier facility at the University of Texas Southwestern Medical Center in Dallas. The generation of Hsd3b6ΔSkin (K14Cre+ / -, Hsd3b6fl / fl) is described below. Mice were housed together in cages of 3-5 mice in all experiments. All mice were housed under a 12-hour light:12-hour dark cycle. Food and water were freely available to the mice according to protocols approved by the Institutional Animal Care and Use Committees (IACUC) at UT Southwestern Medical Center.

[0122] Hsd3b6fl / fl (C57BL6 / J) mice possessing a loxP region surrounding the first coding exon of Hsd3b6 were generated using CRISPR / Cas9 genome editing with a guide RNA targeting the Hsd3b6 locus region (Figure 745 S2A). The guide RNA was injected into fertilized C57BL / 6J embryos at the Children's Research Institute mouse genome engineering facility at UT Southwestern. Healthy blastocysts were transplanted into pseudopregnant mice. The resulting littermates were screened by genome sequencing to detect the insertion of the loxP region. The mice were then homozygous and backcrossed with wild-type C57BL / 6 mice. To generate K14Cre+ / -;Hsd3b6fl / fl(Hsd3b6ΔSkin) mice, Hsd3b6fl / fl mice were crossed with K14Cre+ / - mice 750 times to produce K14Cre+ / -;Hsd3b6fl / + mice. These K14Cre;Hsd3b6fl / + mice were then crossed with Hsd3b6fl / fl mice to obtain experimental mice K14Cre;Hsd3b6fl / fl(Hsd3b6ΔSkin) and the corresponding control, Hsd3b6fl / fl. The status of Hsd3b6fl / fl and Hsd3b6ΔSkin was determined by separation on a 3% agarose gel using PCR primers (Table 1). [Table 1]

[0123] FACS analysis skin Auricular skin from the epidermis of 8-12 week old wild-type, HSD3B6fl / fl, and HSD3B6ΔSkin mice was digested in RPMI medium with 1 mg / mL DNase-I (Sigma-Aldrich, DN25-1G), 0.32 mg / mL liberase-TL (Sigma-Aldrich, 5401020001), and 6 mg / mL collagenase-D (Sigma-Aldrich, 11088858001). The cells were then diced and incubated in a thermocycler at 1400 rpm and 37°C for 1 hour, followed by filtration through a 70 μm cell separation filter. After washing, the cell pellet was suspended in FACS buffer (PBS containing 3% BSA and 2 mM EDTA). The cell suspension was transferred to a 96-well plate with a v-bottom (Corning Inc.: Costar, 3894) and further processed. Cell viability was determined using Ghost-Dye-Red-710 (Cytek, SKU 13-0871-T100). To prevent nonspecific antibody binding, cells were stained with Fc receptor blocking using anti-mouse CD16 / 32 antibody (BD Biosciences, 553142). Different cell populations were evaluated with the following antibodies resuspended in FACS buffer at 4°C for 15 minutes: Brilliant-Violet (BV)-650-anti-CD45 (Biolegend, 103151), FITC-anti-CD3 (Cytek, SKU 35-0032-U025), BV-421-anti-CD11b (Biolegend), and BV711-anti-F4 / 80 (Biolegend). The gating strategy used to determine cell populations was as follows: For all leukocytes, singlet viable cells were gated with the CD45+ marker, from which all T cells were identified as CD45+CD3+ cells, and macrophages were identified as CD45+CD11b+F4 / 80+ cells. Cells were acquired using a NovoCyte flow cytometer and analyzed using NovoExpress software.

[0124] Immunofluorescence microscopy Mouse skin samples were fixed in formalin using the UT Southwestern Histology Core and embedded in paraffin. The samples were deparaffinized with xylene and subsequently rehydrated with decreasing concentrations of ethanol. Heat-induced antigen recovery was achieved in 10 mM sodium citrate buffer. Sections were briefly washed and blocked for 1 hour in blocking / permeabilization buffer (PBS + 5% goat serum + 0.5% Triton® X-100). Sections were incubated overnight in blocking / permeabilization buffer with the following antibodies: anti-HSD3B6 (2.5 μg / mL, Biorbyt, orb592071) and anti-cytokeratin-14 (1 μg / mL, Santa Cruz Biotechnology, sc-53253). After a brief wash with PBST (PBS + 0.2% Tween®-20), sections were incubated with the corresponding secondary antibodies: donkey anti-rabbit alexa-fluor-647 (2 μg / mL, Jackson Immuno, 711-605-152) and donkey anti-mouse alexa-fluor-594 (2 μg / mL, Thermofisher Scientific, A-21203). Slides were then briefly washed with PBST and mounted in a 4,6-diamidino-2-phenylindole (DAPI) mounting medium (Southern Biotech, 0100-20). Images were processed using a ZEISS 780 confocal microscope.

[0125] Bacterial strains and plasmids Staphylococcus aureus strains (Table 1) were streaked onto trypsin-soybean agar (TSA) plates and grown overnight at 37°C. Single colonies were selected and cultured overnight in TSB at 150 RPM and 37°C in a shaking incubator, followed by 1:100 subculturing in a shaking incubator at 37°C to obtain meta-logarithmic bacteria. For fusion reporter strains, all in vitro cultures were performed using TSB in the presence of 10 μg / mL chloramphenicol. Bacteria were pelleted, washed, and re-815 suspended in either TSB for in vitro experiments or PBS for in vivo experiments. HG003, ΔagrC, ΔagrBD, and ΔagrA mutant strains were obtained from Dr. Ferric Fang. Staphylococcus aureus strains from atopic dermatitis skin were obtained from Dr. Julie Segre and Dr. Heidi Kong. All other strains from the Horswill and Harris-Tryon lab collections.

[0126] Construction of a Staphylococcus aureus strain expressing lux The incorporated luxCDABEG cassettes were transduced into Staphylococcus aureus strains HG003, ΔagrBD, and ΔagrC, obtained from Ferric Fang's laboratory, using phage 11, which produces AH6222(lux+), AH6224(lux+), and AH6223(lux+), respectively.

[0127] In vitro luminescence assay Staphylococcus aureus strains expressing Lux (φ11::LL29luxCDABEG) and quorum-sensing lux (pAmiAgrP3lux), HG003, ΔagrBD, and ΔagrC (HG003(AH6225)agr type I, USA100(AH430) type II, and MW2(AH1747)agr type III) (52,65,66), were grown in TSB supplemented with antibiotic selection and subcultured in fresh TSB containing steroid hormones at a TSB 1:200 ratio. The assay was completed in a 96-well, clear-bottom, tissue culture-treated plate with opaque sides and a final well volume of 200 μL. Bioluminescence signals (photons / acquisition time 0.1 sec) were measured using a BioTek H1 Synergy plate reader. The experiment was completed in three consecutive steps, with agr-type specific AIPs AIP-I (Peptide Institute, Inc., catalog number 4515-v), AIP-II (Peptide Institute, Inc., catalog number 4516-v), and AIP-III (Peptide Institute, Inc., catalog number 4517-v) used as positive controls.

[0128] Hemolysis assay The hemolysis assay was completed as previously described, including the following modifications: Overnight cultures of HG003, ΔagrBD, and ΔagrC strains were inoculated at a 1:200 ratio into 10 mL of TSB containing 10 nM testosterone, AIP-I, or vehicle alone. Cells were grown to logarithmic metaphase (OD600 nm 0.6). 1 ml of the culture supernatant was filter-sterilized using a 0.22 μm pore size Millex® sterile syringe filter (catalog no. SLGV033RS). The filtered supernatant, diluted 1:1 with PBS, was added to 25 μL of human blood in a 96-well V-bottom plate and incubated at 37°C for 1 hour with agitation. After spinning at 1000 RPM for 10 minutes, the supernatant was transferred to a flat-bottom 96-well plate. Absorbance was read at 541 nm for hemoglobin using a BioTek H1 Synergy plate reader. Hemolysis % was calculated using the following formula: (A541 of RBC-treated sample - A541 of buffer) / (A541 of H2O - A541 of buffer). Buffer (PBS) = baseline, H2O = 100% hemolysis.

[0129] Neutrophil killing assay Staphylococcus aureus-induced neutrophil killing was measured as previously described. HG003, ΔagrBD, and ΔagrC strains were treated with 10 nM testosterone, AIP-I, or vehicle and grown to logarithmic metaphase (OD600 nm 0.6). Purified human neutrophils (IQ Biosciences) were placed in 96-well plates in 90 μL of RPMI at a rate of 1 × 10⁶ 5Cells were seeded in wells. 10 μL of bacterial supernatant was added (final concentration 10%). After incubation at 37°C and 5% CO2 for 3 hours, the plates were centrifuged at 250 g for 10 minutes. The obtained supernatant was used to measure lactate dehydrogenase (LDH) leakage from damaged cells as a marker of neutrophil lysis using an LDH cytotoxicity detection kit (Invitrogen, catalog no. 2570393). Neutrophil lysis percentage was calculated using neutrophils incubated with 10% RPMI as a 0% lysis control and neutrophils incubated with 0.2% Triton® X-100, defined as 100% lysis.

[0130] Quantitative real-time PCR HG003, USA100 (AH3684), MW2 (AH843), ΔagrBD, ΔagrA, ΔagrC, and atopic dermatitis strains were treated with 10 nM testosterone and / or their respective AIPs and grown to logarithmic metaphase (OD600 nm 0.6). Cells were pelleted and lysed at room temperature using 0.1 mm silica spheres (MP lysis matrix tubes, catalog no. 174701) and lysis matrix B tubes containing lysostafin (Sigma, catalog no. L7386). RNA was purified using the RNeasy Mini Kit (Qiagen, catalog no. 74104). RNA was quantified by absorbance at 260 nm, and its purity was evaluated by the ratio of absorbance at 260 / 280 nm. cDNA was generated using RNA as a template with the High-Capacity Reverse Transcription Kit (Applied Biosystems, catalog number 01071619). Quantitative real-time PCR was performed by amplifying the cDNA using Power SYBR Green Master Mix (Applied Biosystems, catalog number 2749999) and the QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems). Relative expression levels were calculated using the comparative Ct (ΔΔCt) method, and the abundance of the transcript was normalized relative to the abundance of the gyrA transcript. The primer sequences are shown in Table 2. [Table 2-1] [Table 2-2]

[0131] RNA Seq In short, HG003 cultures were grown in triplicate in TSB containing 10 nM testosterone, pregnenolone, or DMSO alone to an optical density of 0.6 at OD600 nm. Cells were harvested and treated with RNA Protect Bacteria Reagent (Qiagen, catalog number 76526). Cells were lysed using lysostafin (Sigma, catalog number L7386), RNA was purified using the RNeasy mini kit (Qiagen, catalog number 74104), and sample quality was confirmed by Bio analyzer (Agilent). Ribosomal RNA was depleted using RiboCop for the Bacterial META Removal Kit (Lexogen). A cDNA library was generated in the University of Michigan Microbiome core using the CORALL RNA-seq Library Prep Kit (Lexogen). Samples were barcoded, pooled, and sequenced using an Illumina HiSeq 2000 sequencer with 125×125 paired-end reads. Raw sequencing reads in fastq format were aligned and annotated to the Staphylococcus aureus NCTC8325 reference genome with annotated sRNA(70) using QiagenCLC Genomics Workbench default settings (version 21.0.5): mismatch cost, 2; insertion and deletion cost, 3; length and similarity rate, 0.8. Normalized and differential expression calculations of uniquely mapped bacterial transcripts were performed using CLC. All transcripts with FDR-adjusted p-values ​​< 0.05 were considered significant.

[0132] Staphylococcus aureus skin infection Prior to the mouse infection study, the mice were adapted to an animal biosafety level 2 (ABSL-2) animal containment facility. Age, strain, and sex-matched C57BL / 6 male and female mice, Hsd3b6fl / fl and Hsd3b6ΔSkin, were used in the study. The mouse model of cutaneous Staphylococcus aureus exposure, as previously described, was followed. Briefly, the dorsal skin of anesthetized mice (2% isoflurane) was shaved and depilated (Nair cream). After 24 hours, bioluminescent Staphylococcus aureus strains were grown to the logarithmic metaphase, pelletized, resuspended in PBS, and 1 × 10⁶ cells were collected. 6 An inoculation containing CFU was obtained. 1 × 10⁻⁶ nanomoles of testosterone, AIP-I, or the same volume of vehicle, or not. 6 A 100 μL volume of PBS containing CFU was placed on a sterile gauze pad, attached to shaved skin with a transparent bio-occlusive bandage (Tegaderm, 3M, Henry Schein Medicals, catalog number 1622W), and immobilized for 4 days with an adhesive bandage (BAND-AID, Johnson and Johnson, American White Cross, catalog number 1275033). Photons emitted from bioluminescent bacteria were collected during automated exposure using an IVIS Lumina3 imager machine and living imaging software (Xenogen, Alameda, CA). Bioluminescent image data is presented as a pseudo-color scale overlaid on grayscale photographic images (blue represents the lowest intensity signal, red represents the highest intensity signal). Using the image analysis tool of the living imaging software, a circular (uniform area) analysis window was superimposed on the dorsal region of the infected area, and the corresponding bioluminescent value (total flux) was measured and plotted against the number of days after infection. Mice were randomly assigned to treatment groups, and at the experimental endpoint, the mice were humanely euthanized using carbon dioxide inhalation.

[0133] Measurement of PSM alpha expression The relative expression of the AgrA-dependent pathogenic factor phenol-soluble modulin alpha (PSMα) in transcripts was quantified by RT-PCR and compared with the expression of the bacterial housekeeping gene DNA gyrase (gyrA). Mean ± SEM values ​​are plotted.

[0134] In vivo experiment Female HSD3B6 ΔSkin Mice were infected percutaneously with the bioluminescent MRSA strain MRSA-lux. The course of infection was tracked in vivo over time using an in vivo imaging system (IVIS) that detects bacterial bioluminescence in 2-D and 3-D.

[0135] Quantitative and statistical analysis Data represent the mean ± standard error of the mean. While formal randomization techniques were not used, mice were randomly assigned to experiments, and samples were processed in any order. Mouse skin samples determined to be in the anagen hair cycle were excluded. All statistical analyses, except for the bioluminescence imaging data analyzed as described above, were performed using GraphPad Prism software. A two-sided Student's t-test was used to assess the statistical significance of differences between two treatments. One-way ANOVA was used to assess the statistical significance of differences between three or more treatments. Outliers within the experiment were identified and removed using Grubb's test. For RNA-seq experiments, expression data were analyzed using CLC. All transcripts with an FDR-adjusted p-value < 0.05 were considered significant.

[0136] Example 1. Ent-testosterone reduced the expression of pathogenic genes in several strains of Staphylococcus aureus. To evaluate the efficacy of ent-testosterone, Staphylococcus aureus strains LAC (type I), USA 100 (type II), MW2 (type III), and MN EV (type IV) were treated with 10 nM ent-testosterone, 10 nM testosterone, or a vehicle. The relative expression of the AgrA-dependent pathogenic factor phenol-soluble modulin alpha (PSMα) was quantified by RT-PCR and compared to the expression of the bacterial housekeeping gene DNA gyrase (gyrA) (Figure 2). Ent-testosterone was found to inhibit PSMα expression in all Staphylococcus aureus type I, II, III, and IV strains, in contrast to testosterone, which enhanced the expression of the pathogenic factor PSMα.

[0137] Example 2: Ent-testosterone reduced quorum sensing in mice in vivo. The effects of ent-testosterone were further tested in vivo. HSD3B6 ΔSkin Female mice were subjected to bioluminescence of MRSA. agr-P3 Transdermal infection was induced with the lux reporter strain and treated with 10 nM ent-testosterone, 10 nM testosterone, or vehicle. Hsd3b6 ΔSkin Mice secrete less testosterone in their skin compared to control mice. The course of infection over time was tracked in vivo using an in vivo imaging system (IVIS) that detects bacterial bioluminescence in 2-D and 3-D. Representative images at 1, 2, 3, and 4 hours after treatment are shown in Figure 3, and mean bioluminescence was plotted hourly for more than 8 hours post-treatment (Figure 4). As shown in Figures 3 and 4, testosterone enhanced bioluminescence, while ent-testosterone inhibited bioluminescence over time. These results indicate that treatment with ent-testosterone inhibited agr-induced bioluminescence, and therefore indicate that ent-testosterone can inhibit quorum sensing.

[0138] Example 3: Ent-testosterone dosage Furthermore, various doses of Ent-testosterone were tested in vitro using the MRSA agr-P3 lux reporter strain of MRSA. MRSA agr-P3 lux strains were treated with 10 nM, 100 nM, 1 μM, and 10 μM of Ent-testosterone, testosterone, AIPII, or vehicle, and bioluminescence was quantified over time as shown in Figure 5 and Figure 6. At all doses tested, Ent-testosterone reduced bioluminescence, indicating that Ent-testosterone inhibited MRSA.

[0139] Example 4: Enantiomer of testosterone inhibits the pathogenesis of Staphylococcus aureus To understand the specificity of the interaction between testosterone and the agr system, the effect of ent-testosterone (ent-T), a stereoisomer of testosterone, on the pathogenesis of Staphylococcus aureus was tested. Like other non-natural enantiomers, ent-T has the same physicochemical properties as testosterone, but has mirror image orientation at all six chiral centers, resulting in a change in the rotation of polarized light (Figure 7A). Enantiomers have been observed to possess unique receptor binding and signaling properties. Unlike other tested hormones that showed a neutral effect on the agr system (Figure 9A to Figure 9D), Ent-testosterone acted as a broad-spectrum inhibitor of the agr quorum sensing system (Figure 2, Figure 7B to Figure 7D, Figure 8A to Figure 8B). Ent-testosterone was able to reduce Staphylococcus aureus-dependent hemolysis and neutrophil killing in type I agr strains (Figure 7B to Figure 7C). Furthermore, Ent-testosterone had the ability to decrease the expression of agr transcriptional readout in type II, type III, and atopic dermatitis strains of Staphylococcus aureus (Figure 7B to Figure 7D, Figure 8A). Moreover, topical application of Ent-testosterone to the skin of mice infected with Staphylococcus aureus turns off quorum sensing in vivo (Figure 3, Figure 4, Figure 8B). Thus, ent-T is a global inhibitor of Staphylococcus aureus quorum sensing that functions across agr types, similar to established bacterial-derived regulators of agr such as non-cognate AIP (Figure 7B to Figure 7C).

[0140] Example 5: Ent-testosterone mediates quorum quenching. The effects of ENT-testosterone on an infection model were tested by disrupting the skin barrier of mice using tape stripping. A previously established skin infection protocol was used. Briefly, mice were anesthetized with isoflurane and tape stripping was performed (TEWL = 20 g / m²). 2 / h), after 24 hours, 10 in 100 μl 7 CFU MRSA SAP 430 was applied to the dorsal skin and spread using a swab. The inoculation was allowed to dry for 10 minutes, and the mice were returned to their cages. The treatment was performed once daily. Tegaderm was not used during the treatment. In vivo, no difference was observed between the control vehicle-treated and ENT-treated mice with MRSA. However, the TEWL-treated group showed a significant difference compared to the control vehicle-treated group (Figures 10A-10B). This may be because Ent-testosterone mediates quorum quenching at the site of infection in vivo. This inhibits the production of AIP, a member of the family of signaling molecules used in quorum sensing. Therefore, proposed Ent-testosterone may have the potential to reduce AD ​​symptoms such as itching, redness, swelling, pain, and / or skin barrier disruption by inhibiting quorum sensing and thereby restoring the barrier without eradicating Staphylococcus aureus, which is part of the natural skin microbiome.

[0141] Example 6: Ent-testosterone inhibits quorum sensing in vivo. ENT-testosterone was applied epicutively to male mice at 10 and 100 nanomoles, followed immediately by application of the AH2659 agrP3 reporter strain, and agrkinetics were recorded every hour. This followed a previously described mouse model of cutaneous Staphylococcus aureus exposure. Briefly, the dorsal skin of anesthetized mice (2% isoflurane) was shaved and depilated (Nair cream). After 24 hours, the bioluminescent Staphylococcus aureus strain was grown to logarithmic metaphase, pelletized, resuspended in PBS, and recorded at 1 × 10⁶ 6 An inoculation containing CFU was obtained. 1 × 10 6 A 100 μL volume of PBS containing CFU was placed on a sterile gauze pad, attached to shaved skin with a transparent bio-occlusive bandage (Tegaderm, 3M, Henry Schein Medicals, catalog no. 1622W), and secured with an adhesive bandage (BAND-AID, Johnson and Johnson, American White Cross, catalog no. 1275033). Photons emitted from the bioluminescent bacteria were collected during automated exposure using an IVIS Lumina3 imager machine and living imaging software (Xenogen, Alameda, CA).

[0142] As shown in Figures 11A and 11B, ENT-testosterone was found to inhibit quorum sensing. No weight changes were observed between the treatment groups on the day of infection (Figure 11C). There were also no differences in the applied TEWL between the treatment groups (Figure 11D).

[0143] Furthermore, to test whether high doses of ENT-testosterone are superior to testosterone on the skin, experiments were conducted using gradually increasing concentrations of ENT-testosterone in combination with testosterone, and agrP3 brightness was measured. As shown in Figure 12, it was found that increasing the concentration of ENT-testosterone, in the presence of testosterone, reduced HG003 agrP3 brightness in a dose-dependent manner.

[0144] Summary of Examples In summary, ent-testosterone inhibited the expression of pathogenic factors and quorum sensing both in vitro and in vivo. These results indicate that ent-testosterone can inhibit bacterial pathogenicity and thus has therapeutic applications in the treatment of bacterial infections or conditions complicated by bacterial colonization.

[0145] In contrast to progesterone and estradiol, which had a neutral effect on Staphylococcus aureus, ENT-testosterone, a stereoisomer of testosterone, reduced quorum sensing and decreased bacterial-induced hemolysis and neutrophil killing. ENT-testosterone also inhibited quorum sensing in vivo during Staphylococcus aureus infection. These findings demonstrate that Staphylococcus aureus responds to testosterone in an enantioselective manner, with testosterone generating an activation signal and its chiral counterpart, ENT-testosterone, resulting in the same phenotypic inhibition. Since natural hormones and their enantiomers have been shown to have the same physicochemical properties within the membrane, the distinct phenotypes of testosterone and ENT-testosterone suggest that the effects of these hormones on agr signaling are independent of any predicted effects of androgenic hormones on the bacterial membrane.

Claims

1. A composition for the treatment of bacterial infections in subjects requiring treatment for bacterial infections, wherein the composition comprises enantiomer testosterone (Ent-testosterone).

2. A method for treating a subject having or at risk of having a bacterial infection, wherein the method comprises administering a composition comprising ent-testosterone to the subject.

3. A method for treating a subject having or at risk of having a bacterial skin and soft tissue infection (SSTI), wherein the method comprises administering a composition comprising ent-testosterone to the subject.

4. A method for treating a subject having or at risk of having a condition complicated by bacterial colonization, wherein the method comprises administering a composition containing ent-testosterone to the subject.

5. The method according to claim 4, wherein the condition is atopic dermatitis, folliculitis, pemphigus, bullous pemphigoid, or cutaneous T-cell lymphoma.

6. The composition or method according to any one of claims 1 to 5, wherein the bacterial infection is caused by bacteria of the genus Staphylococcus.

7. The composition or method according to claim 6, wherein the bacterial infection is caused by Staphylococcus aureus or Staphylococcus epidermidis.

8. The composition or method according to claim 7, wherein the Staphylococcus aureus is methicillin-resistant Staphylococcus aureus (MRSA), methicillin-sensitive Staphylococcus aureus (MSSA), vancomycin-intermediate resistant Staphylococcus aureus (VISA), and / or vancomycin-resistant Staphylococcus aureus (VRSA).

9. The composition or method according to claim 7, wherein the Staphylococcus aureus is Staphylococcus aureus strain LAC, Staphylococcus aureus strain USA 100, Staphylococcus aureus strain MW2, and / or Staphylococcus aureus strain MN EV.

10. The composition or method according to any one of claims 1 to 9, wherein the subject is a mammal.

11. The composition or method according to claim 10, wherein the subject is a human.

12. The composition or method according to any one of claims 1 to 11, wherein the ent-testosterone (a) reduces the expression of pathogenicity factors or pathogenicity-related protein genes by at least 10% compared to an untreated control, (b) reduces staphylococcus-induced hemolysis by at least 10%, and / or (c) reduces staphylococcus-induced neutrophil killing by at least 5%.

13. The composition or method according to claim 12, wherein the pathogenicity-related protein gene is an accessory gene regulator (agr) type I.

14. The composition or method according to claim 12, wherein the pathogenicity-associated protein gene is of type agr II.

15. The composition or method according to claim 12, wherein the pathogenicity-associated protein gene is of type agr III.

16. The composition or method according to claim 12, wherein the pathogenicity-related protein gene is of type agr IV.

17. The composition or method according to any one of claims 1 to 16, wherein the ent-testosterone reduces quorum sensing by the bacteria by at least 10% compared to an untreated control.

18. The composition or method according to any one of claims 1 to 17, wherein the ent-testosterone is in a concentration of about 10 nM to about 10 μM.

19. The composition or method according to any one of claims 1 to 12, wherein the ent-testosterone reduces accessory gene regulator C histidine kinase (agrC).

20. The composition or method according to any one of claims 1 to 19, wherein the composition further comprises a pharmaceutically acceptable carrier.

21. The composition or method according to claim 20, wherein the pharmaceutically acceptable carrier is petrolatum.

22. A method for limiting damage to a target tissue caused by bacterial pathogenicity-associated proteins, wherein the method comprises administering a composition comprising ent-testosterone to the target.

23. The method according to claim 22, wherein the tissue is skin tissue.

24. A method for weakening the pathogenicity of a Staphylococcus species, wherein the method comprises contacting the Staphylococcus species with a composition containing an effective amount of ent-testosterone.

25. The method according to claim 24, wherein the Staphylococcus species is Staphylococcus aureus or Staphylococcus epidermidis.

26. The method according to any one of claims 24 to 25, wherein the Staphylococcus aureus is methicillin-resistant Staphylococcus aureus (MRSA), methicillin-sensitive Staphylococcus aureus (MSSA), vancomycin-intermediate resistant Staphylococcus aureus (VISA), and / or vancomycin-resistant Staphylococcus aureus (VRSA).

27. The method according to any one of claims 24 to 26, wherein the Staphylococcus aureus is Staphylococcus aureus strain LAC, Staphylococcus aureus strain USA 100, Staphylococcus aureus strain MW2, and / or Staphylococcus aureus strain MN EV.

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