ENT-testosterone compositions and methods for treating bacterial infections

Abstract

The present disclosure relates generally to compositions and methods using ent- testosterone. The disclosed ent-testosterone can be used to inhibit quorum sensing of bacteria. The present disclosure further relates to compositions and methods of treating bacterial infections or having a complicated condition due to bacterial colonization.

Description

Cross-references to related applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 552,459, filed February 12, 2024, entitled “ENT-Testosterone Composition and Method for Treating Bacterial Infections”, and U.S. Provisional Patent Application No. 63 / 580,813, filed September 6, 2023, entitled “ENT-Testosterone Composition and Method for Treating Bacterial Infections”, the entire contents of which are incorporated herein by reference.

[0002] Merging of sequence lists This application contains a sequence list, which has been submitted to the Patent Centre in .XML format, the entire contents of which are incorporated herein by reference. The WIPO sequence list was created on August 21, 2024, and the XML copy is named 106546-816096-4247_Sequence listing.xml and is 19 kilobytes in size. Technical Field

[0003] This invention relates to enantiomer-testosterone (ENT-testosterone), and compositions and methods for treating bacterial infections using ENT-testosterone. Background Technology

[0004] Staphylococcus aureus ( S. aureus Skin and soft tissue infections caused by Staphylococcus aureus (SSA) pose a significant public health threat. 7-10% of hospitalized patients suffer from skin and soft tissue infections, and skin infections are the third most common cause of emergency room visits. 75% of these skin infections are caused by Staphylococcus aureus. The emergence of antibiotic-resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA) further exacerbates the disease burden caused by these pathogens. MRSA is the most common causative agent of skin and soft tissue infections in emergency settings and a frequent cause of hospital-acquired infections. Furthermore, MRSA infections result in a total societal cost exceeding $1 billion annually.

[0005] Staphylococcus aureus is an opportunistic pathogen that can exist in a quiescent colony or cause lethal infections. Staphylococcus aureus encodes a large number of virulence factors, including cell surface adhesins and extracellular matrix degrading enzymes, enabling it to bind to, invade, and damage epithelial cells, thus causing infection. The most thoroughly described virulence regulatory system is the helper 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 through a stepwise process: first, the propeptide AgrD is produced in the cytoplasm; subsequently, the propeptide AgrD is processed into AIP by the intracellular peptidase AgrB and secreted extracellularly. Studies have also shown that MroQ is involved in the biosynthetic pathway of AIP. When bacteria reach a sufficient density and the AIP concentration is sufficiently high (“quorum”), AIP binds to and activates histidine kinase AgrC. Upon activation, AgrC phosphorylates the response regulatory protein AgrA; phosphorylated AgrA then automatically induces the expression of agr-related elements and RNAIII transcripts. RNAIII is the main effector of this system, triggering the massive production of Staphylococcus aureus toxins and extracellular enzymes. Besides responding to AIP, AgrC can also respond to other environmental conditions; recent studies have shown that nitric oxide can inhibit Staphylococcus aureus by directly inhibiting AgrC. However, host-derived factors can drive Staphylococcus aureus skin infections in vivo and affect bacterial pathogenicity and the progression of skin infections and diseases. Due to the limited treatment options for bacterial skin infections (e.g., those caused by Staphylococcus aureus), there remains an unmet need to develop therapeutics for the treatment of skin and soft tissue infections. Summary of the Invention

[0006] In one aspect, this disclosure covers a composition for treating a bacterial infection in a subject of need, the composition comprising an enantiomer of testosterone. In some aspects, the composition further comprises a pharmaceutically acceptable carrier. In some aspects, the pharmaceutically acceptable carrier is petrolatum.

[0007] In a further aspect, this article provides a method for treating a subject who has a bacterial infection or is at risk of having a bacterial infection, the method comprising administering to the subject a composition containing Ent-testosterone.

[0008] This disclosure also covers a method of treating a subject who has a bacterial skin and soft tissue infection (SSTI) or is at risk of having a bacterial skin and soft tissue infection (SSTI), the method comprising administering to the subject a composition comprising Ent-testosterone.

[0009] In some aspects, this disclosure includes a method for treating a subject suffering from or at risk of a complicated condition caused by bacterial colonization, the method comprising administering to the subject a composition comprising Ent-testosterone. This document also provides a method for limiting damage to a subject's tissue caused by bacterial virulence-associated proteins, the method comprising administering to the subject a composition comprising Ent-testosterone. In these aspects, the tissue is skin tissue.

[0010] This disclosure also covers a method for reducing the virulence of Staphylococcus spp. bacteria, the method comprising contacting the Staphylococcus spp. bacteria with a composition containing an effective amount of Ent-testosterone.

[0011] In some respects, the condition is atopic dermatitis, folliculitis, pemphigus, bullous pemphigoid, or cutaneous T-cell lymphoma.

[0012] In some cases, bacterial infections are caused by bacteria of the genus *Staphylococcus*. In others, they are caused by *Staphylococcus aureus*. In some cases, *Staphylococcus aureus* is methicillin-resistant *Staphylococcus aureus* (MRSA), methicillin-sensitive *Staphylococcus aureus* (MSSA), vancomycin-intermediate *Staphylococcus aureus* (VISA), or vancomycin-resistant *Staphylococcus aureus* (VRSA). In some cases, *Staphylococcus aureus* is *Staphylococcus aureus* LAC strain, *Staphylococcus aureus* USA 100 strain, *Staphylococcus aureus* MW2 strain, or *Staphylococcus aureus* MN EV strain. In other cases, bacterial infections are caused by *Staphylococcus epidermidis*.

[0013] In some respects, the subjects were mammals. In other respects, the subjects were humans.

[0014] In some respects, compared with untreated control subjects, Ent-testosterone reduced the expression of virulence factors or virulence-related protein genes by at least 10%. In some respects, the virulence-related protein gene is agr type I. In some respects, the virulence-related protein gene is agr type II. In some respects, the virulence-related protein gene is agr type III. In some respects, the virulence-related protein gene is agr type IV. In a further respects, Ent-testosterone reduced Staphylococcus-induced hemolysis by at least 10%; and / or reduced Staphylococcus-induced neutrophil killing by at least 5%.

[0015] In some respects, Ent-testosterone reduced bacterial quorum sensing by at least 10% compared to untreated control subjects. In some respects, the effective amount of Ent-testosterone is approximately 10 nM to 10 µM. In some respects, the effective amount of Ent-testosterone reduced the helper gene regulator C histidine kinase (agrC).

[0016] Other technical features can be readily understood by those skilled in the art through the following drawings, description and claims. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the agr system in Staphylococcus aureus that coordinates the expression of virulence factors.

[0018] Figure 2 The relative expression levels of PSMα 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 solvent are shown (RT-PCR quantification). Data are plotted as mean ± standard error (error bars). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns indicates no significant difference in a two-tailed unpaired t-test.

[0019] Figure 3 This study demonstrates the effects of percutaneous bioluminescent MRSA infection at 1, 2, 3, and 4 hours after treatment with 10 nM Ent-testosterone, Ent-testosterone, or solvent. agr-P3 HSD3B6 of the lux reporter strain Δ皮肤 Representative images of female mice. Wild-type female mice were infected via epidermal transdermal administration of 1×10⁻⁶ cells. 6 CFU of *Staphylococcus aureus* reporter strain agr-P3 was used, and the strain was treated with either testosterone or Ent-testosterone. Changes in bioluminescence over time were quantified. The study included testosterone group (n=4), Ent-testosterone group (n=4), and solvent group (n=4).

[0020] Figure 4 This study demonstrated the effects of percutaneous bioluminescent MRSA infection 1 to 8 hours after treatment with 10 nM Ent-testosterone, 10 nM testosterone, or solvent. agr-P3 HSD3B6 of the lux reporter strain Δ皮肤 Mean bioluminescence of female mice. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.05, determined by unpaired t-test.

[0021] Figure 5 MRSA is shown agr-P3Quantitative bioluminescence of lux strains over time under treatment with 10 nM, 100 nM, 1 µM and 10 µM Ent-testosterone, AIPII or solvent.

[0022] Figure 6 MRSA was displayed agr-P3 Quantitative bioluminescence of lux strains over time under treatment with 10 nM, 100 nM, 1 μM and 10 μM Ent-testosterone or testosterone.

[0023] Figures 7A-7D This study demonstrated that Ent-testosterone inhibits Staphylococcus aureus virulence and agr quorum sensing. Figure 7A The structures of testosterone and its stereoisomer Ent-testosterone (ent-T) are shown. Figures 7B-7C Wild-type Staphylococcus aureus 695 (HG003) is shown, treated with 100 nM ent-T, AIP-II or untreated (solvent control). Figure 7B The percentage of hemolysis induced by Staphylococcus aureus is shown. Figure 7C The percentage of neutrophil killing induced by Staphylococcus aureus (C) is shown (n=3). Figure 7D The qRT-PCR expression levels of the psmα gene in *Staphylococcus aureus* strains treated with 10 nM testosterone or 10 nM ent-T are shown. Relative psmα expression levels were normalized relative to the expression level of the housekeeping gene gyrA. Data are plotted as mean ± standard error (error bars). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns indicates no significant difference in a two-tailed unpaired t-test.

[0024] Figures 8A-8B This study demonstrated that enantiomeric testosterone (ent-T) inhibits the expression of Staphylococcus aureus virulence factors and quorum sensing. Figure 8A The results of qRT-PCR for Staphylococcus aureus strains from patients with atopic dermatitis after 10 nM ent-T treatment are shown (n=3). The expression levels of the target gene RNAIII were normalized to the expression levels of the housekeeping gene gyrA. Data are plotted as mean ± standard error (error bars). *p<0.05, two-tailed unpaired t-test. Figure 8B It shows Figure 4 Representative bioluminescent images, i.e., transepidermal infection of 1×10 6 Female wild-type mice with CFU agr-P3 Staphylococcus aureus reporter strain, treated with testosterone, ent-T, solvent, or untreated (n=4).

[0025] Figure 9AFigure 9D shows the readout of testosterone and DHT activating quorum sensing in Staphylococcus aureus, but other hormone classes cannot activate quorum sensing. Figure 9A The in vitro bioluminescence of the agr reporter strain (HG003 agrP3::lux) is shown after treatment with 10 nM testosterone, DHT, progesterone, or estradiol. Figure 9B The in vitro bioluminescence of the agr reporter strain after DHT treatment is shown (HG003 agrP3::lux). Figure 9C The in vitro bioluminescence of the agr reporter strain after progesterone treatment is shown (HG003 agrP3::lux). Figure 9D The in vitro bioluminescence of the agr reporter gene after treatment with AIP-I or estradiol is shown (HG003 agrP3::lux).

[0026] Figure 10A A series of photographs of mice after hair removal with tape and MRSA inoculation on days 0, 1, and 2.

[0027] Figure 10B The graphs are a series of line graphs, with the top graph showing the bioluminescence of MRSA in vivo and the bottom graph showing the effect of MRSA on TEWL (transdermal water loss) in vivo, which is a marker of skin barrier function.

[0028] Figure 11A Images of a series of male mice show the luminescence intensity of agrP3 at 0, 1, 2, 3, 4, 5, 6, 7 and 8 hours after treatment with the AH2659 agrP3 reporter strain, followed by treatment with solvent, 10 nanomolar Ent-testosterone or 100 nanomolar Ent-testosterone.

[0029] Figure 11B The graph shows the changes in agrP3 luminescence intensity at 0, 1, 2, 3, 4, 5, 6, 7 and 8 hours after treatment with the AH2659 agrP3 reporter strain, followed by treatment with solvent, 10 nanomolar Ent-testosterone or 100 nanomolar Ent-testosterone.

[0030] Figure 11C The bar chart shows the average body weight of mice in different treatment groups on the day of infection with the AH2659 agrP3 reporter strain.

[0031] Figure 11D The plot shows the TEWL of different treatment groups on the day of infection with the AH2659 agrP3 reporter strain.

[0032] Figure 12The graph is a line graph showing the change in agrP3 luminescence intensity over time with increasing Ent-testosterone concentration, with or without testosterone.

[0033] The accompanying drawings are not intended to limit this disclosure to the specific embodiments disclosed and described herein. The drawings are not necessarily drawn to scale, and their purpose is to clearly illustrate the principles of certain embodiments of this disclosure. Detailed Implementation

[0034] The following detailed description refers to the accompanying drawings, which illustrate various aspects of this disclosure. The drawings and description are intended to describe various aspects of this disclosure in sufficient detail to enable those skilled in the art to implement it. Other components and changes may be employed without departing from the scope of this disclosure. Therefore, the following description should not be construed as limiting.

[0035] This disclosure is partly based on the discovery that enantiomer testosterone (ENT-testosterone) can inhibit bacteria and can be used as a therapeutic agent for bacterial infections or complicated conditions caused by bacterial infections. ENT-testosterone has been found to inhibit the expression of virulence factors in Staphylococcus types I, II, III, and IV. Furthermore, it has been found that ENT-testosterone can reduce quorum sensing by decreasing the activity of the helper gene regulator C histidine kinase (agrC). This disclosure also provides compositions and methods for treating or preventing bacterial infections or complicated conditions caused by bacterial infections using ENT-testosterone.

[0036] I. Terminology To facilitate understanding of the principles of this disclosure, reference is made to preferred aspects and specific language is used in the description. Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains.

[0037] As used in this specification, the articles "a" and "an" refer to one or more (i.e., at least one) of the grammatical objects. For example, "an element" means at least one element, but may also include multiple elements.

[0038] The word "approximately" is used to provide flexibility at the endpoints of a numerical range, allowing a given value to be "slightly above" or "slightly below" the endpoints without affecting the expected result. When used with a numerical value, "approximately" indicates that the value can vary within plus or minus 5% of its value or less.

[0039] Throughout this specification, unless the context otherwise requires, the terms "comprising" and "including" shall be understood to include the said components, features, elements or steps, or groups of components, features, elements or steps, but do not exclude any other integers, steps or groups of integers or steps.

[0040] As used herein, "and / or" refers to and covers any and all possible combinations of one or more of the listed items, as well as the case where there is no combination under alternative interpretation ("or").

[0041] In this specification, the transitional phrase "consistently of..." (and its grammatical variations) should be interpreted as encompassing the listed materials or steps "as well as those materials or steps that do not substantially affect the essential and novel features of the claimed invention". Therefore, "consistently of..." as used herein should not be construed as equivalent to "comprising".

[0042] Furthermore, this disclosure also contemplates that in some embodiments, any feature or combination of features described herein may be excluded or omitted. For example, if the specification states that a complex comprises components A, B, and C, it expressly means that any one of A, B, or C, or any combination thereof, may be omitted and abandoned, either individually or in any combination.

[0043] The numerical ranges listed herein are for illustrative purposes only and are intended to refer to each individual value within that range (unless otherwise stated), and each individual value is incorporated into the specification as if listed separately. For example, if the concentration range is stated as 1% to 50%, it is intended that ranges such as 2% to 40%, 10% to 30%, or 1% to 3% are explicitly listed in this specification. The above are merely examples of specific intent, and all possible combinations of values ​​between the listed minimum and maximum values, including the endpoints, should be considered as explicitly stated in this specification.

[0044] As used herein, “treatment,” “therapeutic approach,” and / or “treatment regimen” refers to a clinical intervention for a patient’s disease, condition, or physical condition, or for which the patient may be susceptible. Treatment goals include relieving or preventing symptoms, delaying or halting the progression or worsening of the disease, condition, or physical condition, and / or achieving remission of the disease, condition, or physical condition.

[0045] As used in this article, “prevention” means eliminating or delaying the onset of a particular disease, condition or illness, or reducing its severity relative to the timing, severity and / or severity of the onset without intervention.

[0046] The term "effective dose" or "therapeutic effective dose" refers to a dose sufficient to produce beneficial or desirable biological and / or clinical effects.

[0047] As used herein, the terms “individual,” “subject,” “host,” and “patient” are used interchangeably and refer to any mammalian subject requiring diagnosis, treatment, prevention, or therapy, such as a human, pet, livestock, horse, or other animal. As used herein, the terms “subject” and “patient” are used interchangeably and refer to both human and non-human animals. The term “non-human animal” in this disclosure encompasses all vertebrates, including mammals and non-mammals such as non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, etc. In one aspect, the subject may be a rodent, such as a mouse, rat, guinea pig, etc. In another aspect, the subject may be livestock. Suitable, non-limiting examples of livestock include pigs, cattle, horses, goats, sheep, ostriches, and alpacas. In yet another aspect, the subject may be a companion animal. Non-limiting examples of companion animals include pets, such as dogs, cats, rabbits, birds, etc. In yet another aspect, the subject may be a zoo animal. As used in this article, "zoo animals" refers to animals that can be seen in zoos, including non-human primates, large felines, wolves, and bears. In some respects, the subjects may be humans; in others, they may be people requiring treatment for bacterial infections.

[0048] As used in this article, "quorum sensing" refers to bacterial signal transduction and response. Quorum sensing enables bacteria to sense their own population size and regulate gene expression based on population density. Quorum sensing is a cell density-dependent gene regulation involving freely diffusing molecules synthesized by the cell, namely self-inducing agents or self-inducing peptides (AIPs).

[0049] As used in this article, "helper gene regulator" or "agr" refers to a global regulator of virulence. Figure 1 As shown, the agr system activates the receptor histidine kinase AgrC on the bacterial membrane via a secreted small self-inducible peptide (AIP). AgrC phosphorylates the transcription factor AgrA, which in turn activates the transcription of the P2 and P3 promoters in the operon. Activation of P3 drives the synthesis of the operon effector RNAIII, which regulates the expression of over 200 virulence genes associated with invasive infection. Staphylococcus aureus isolates possess one of four agr alleles (type I, II, III, or IV), each encoding a secretion-specific AIP factor (i.e., AIP1, AIP2, AIP3, or AIP4) recognized by the corresponding AgrC histidine kinase. Each Staphylococcus aureus isolate contains only a single copy of the agr system, and each specific species produces different types of AIP signals through variations in the agrBDCA operon. Staphylococcus aureus isolates carry one of these four alleles, and isolates carrying any of these alleles can cause human disease.

[0050] As used herein, "enantiomer-testosterone," "Ent-testosterone," or "Ent-17α-hydroxyandrost-4-en-3-one" are used interchangeably to refer to the enantiomers of testosterone, the primary male sex hormone. Enantiomers are paired stereoisomers, also known as antipodes, optical enantiomers, or optical isomers. They are mirror images of each other and cannot be superimposed. Enantiomers have the same atomic configuration but opposite three-dimensional conformations. Testosterone is a steroid hormone primarily produced by the interstitial cells of the male testes, with small amounts also produced by the female ovaries. Small amounts of this hormone are also synthesized in the adrenal cortex of both men and women.

[0051] As used herein, “expression,” “level,” or “expression level” refers to the amount of a specific protein present in a sample. This amount can be a numerical value, a proportion, or a percentage of the analyte relative to a control sample, and can be either an absolute or relative amount.

[0052] As used herein, "skin and soft tissue infection" or "SSTI" refers to a bacterial infection of the skin, muscles and ligaments, tendons, and other connective tissues. Non-limiting examples of SSTI include: abscesses, cellulitis, folliculitis, MRSA (methicillin-resistant Staphylococcus aureus) infection, Staphylococcus (Staphylococcus aureus) infection, erysipelas, impetigo, boils, and carbuncles.

[0053] Biological samples may be taken from any biological tissue, body fluid, or cells of the subject, and may be in solid or liquid form, or may be heterogeneous cell populations. Non-limiting examples of suitable biological samples include sputum, serum, blood, blood cells (such as leukocytes), biopsy tissue, urine, peritoneal fluid, pleural fluid, and their derived cells. Biopsy types may include fine-needle aspiration biopsy, core needle biopsy, vacuum-assisted biopsy, open surgical biopsy, curettage biopsy, drill biopsy, excisional biopsy, curettage biopsy, or deep curettage biopsy. Biological samples may also contain tissue sections, such as frozen sections or formalin-fixed sections for histological examination. Samples may be skin tissue, infected surrounding tissue, or non-infected tissue. Methods for collecting biological samples from subjects are well known in the art.

[0054] Samples may be collected from the subject once or multiple times before, during, and / or after diagnosis. In some respects, samples may be collected before, during, and / or after treatment for the bacterial infection. In other respects, samples may be collected before, during, and / or after the administration of the treatment regimen. Furthermore, after the initial sample collection, samples may be collected repeatedly at multiple stages.

[0055] In some respects, the control sample may be obtained from healthy subjects. In some respects, the control sample may contain uninfected cells. In some respects, the uninfected cells may originate from the same tissue type as the infected cells. For example, if the infected cells originate from skin, the uninfected cells may be taken from healthy skin tissue. In some respects, the control may comprise the average level of a subject's pre-infection sample. In some respects, the control sample may be a sample of the subject prior to diagnosis or treatment. In some respects, the control is one or more individuals with similar characteristics to the infected subject. In some respects, the control may be the average of a combination of different healthy sources (e.g., multiple healthy control subjects). In some respects, the control sample may be a pooled sample.

[0056] As used herein, "effective amount" can refer to an amount sufficient to achieve the desired effect, such as in the treatment and / or prevention of a disease or condition (e.g., bacterial infection). As used herein, "effective amount" can refer to an amount sufficient to produce the desired effect on an adverse condition (e.g., bacterial infection).

[0057] In one aspect, "therapeutic effective dose" refers to the amount of the disclosed composition used to: (i) treat a disease or condition (e.g., a bacterial infection); (ii) reduce, alleviate, or eliminate one or more symptoms associated with a disease or condition (e.g., a bacterial infection); or (iii) delay the onset of one or more symptoms of a disease or condition (e.g., a bacterial infection). The specific therapeutic effective dose level for a particular patient depends on a variety of factors, including the type of bacterial infection being treated; the disclosed composition used; the method of the present disclosure used; the patient's age, weight, overall health condition, sex, and diet; the time of administration; the route of administration; the excretion rate 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 known in the medical field. For example, those skilled in the art are fully capable of setting the initial dose of the disclosed composition at a level below the dose required to achieve the desired therapeutic effect and gradually increasing the dose until the desired effect is achieved. If necessary, the effective daily dose can be divided into multiple administrations. Therefore, a single dose of the disclosed composition, the disclosed pharmaceutical formulation, the disclosed therapeutic agent, or a combination thereof may contain the aforementioned amount or subunits constituting a daily dose. If any contraindications exist, the dosage may be adjusted by the attending physician. Dosage may vary and may be administered once or multiple times daily for one or several days. Appropriate dosage guidelines for specific classes of drug formulations are documented in relevant literature. In many other respects, the formulation may be administered at a "preventatively effective dose" (i.e., the amount that effectively prevents signs or symptoms associated with a disease or condition, such as a bacterial infection).

[0058] II. Ent-testosterone This disclosure covers Ent-testosterone, which can inhibit bacteria. Ent-testosterone can inhibit the expression of virulence factors in pathogenic microorganisms, such as Staphylococcus spp.

[0059] In some respects, the Ent-testosterone disclosed herein has the following chemical structure: In some respects, Ent-testosterone can block, inhibit, or reduce agrC activity. In some respects, the Ent-testosterone described herein can reduce agrC activity compared to agrC activity in the absence of Ent-testosterone described herein. In some respects, 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 agrC activity in the absence of Ent-testosterone described herein.

[0060] In some respects, Ent-testosterone can block, inhibit, or reduce the expression of one or more virulence-related protein genes or virulence factors in bacteria. In some respects, virulence-related protein genes belong to helper gene regulator (agr) type I. In some respects, virulence-related protein genes belong to helper gene regulator (agr) type II. In some respects, virulence-related protein genes belong to helper gene regulator (agr) type III. In some respects, virulence-related protein genes belong to helper gene regulator (agr) type IV. In some respects, the Ent-testosterone described herein can reduce the expression of one or more virulence factors in bacteria compared to bacteria without the Ent-testosterone described herein. In some respects, the Ent-testosterone described herein can reduce the expression of one or more virulence 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 bacteria in which the Ent-testosterone described herein is absent. In some respects, the Ent-testosterone described herein can reduce the expression of one or more virulence factors in bacteria by at least about 10%.

[0061] In some respects, Ent-testosterone can block, inhibit, or reduce bacterial quorum sensing. In some respects, the Ent-testosterone described herein can reduce bacterial quorum sensing levels compared to bacteria without Ent-testosterone described herein. In some respects, 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 bacteria without Ent-testosterone described herein. In some respects, the Ent-testosterone described herein can reduce bacterial quorum sensing by at least about 10%.

[0062] In some respects, Ent-testosterone can block, inhibit, or reduce bacterial growth. In some respects, the Ent-testosterone described herein can reduce bacterial growth compared to bacterial growth without Ent-testosterone described herein. In some respects, 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 without Ent-testosterone described herein. In some respects, the Ent-testosterone described herein can reduce bacterial growth by at least about 10%.

[0063] In some respects, Ent-testosterone can block, inhibit, or reduce bacterial infections. In some respects, the Ent-testosterone described herein reduces bacterial infections compared to bacterial infections without Ent-testosterone described herein. In some respects, the Ent-testosterone described herein reduces bacterial infections by approximately 5% to approximately 99% (e.g., approximately 5%, approximately 10%, approximately 15%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 95%, approximately 99%) compared to bacterial infections without Ent-testosterone described herein. In some respects, the Ent-testosterone described herein reduces bacterial infections by at least approximately 10%.

[0064] In some respects, the bacterium can be any bacterium with quorum sensing. In some respects, the bacterium can be any bacterium with an agrC signaling system. In some respects, the bacterium is a Staphylococcus spp. In some respects, the bacterium is Staphylococcus aureus or Staphylococcus epidermidis. In some respects, Staphylococcus aureus can be methicillin-resistant Staphylococcus aureus (MRSA), methicillin-sensitive Staphylococcus aureus (MSSA), vancomycin-intermediate Staphylococcus aureus (VISA), or vancomycin-resistant Staphylococcus aureus (VRSA). In some respects, Staphylococcus aureus is Staphylococcus aureus LAC strain, Staphylococcus aureus USA 100 strain, Staphylococcus aureus MW2 strain, or Staphylococcus aureus MN EV strain.

[0065] III. Composition In some aspects, the present invention provides compositions comprising Ent-testosterone. The compositions disclosed herein may be pharmaceutical compositions. In some aspects, the pharmaceutical compositions described herein may comprise Ent-testosterone and at least one pharmaceutically acceptable carrier and / or excipient. Pharmaceutically acceptable carriers or excipients suitable for use in the compositions described herein are well known to those skilled in the art for the preservation and delivery of active ingredients (such as hormones) to any mammalian subject (including humans and other mammals).

[0066] In some respects, the compositions described herein may be administered to subjects in need. In some respects, the subject is a person. In some respects, the subject is a subject with a bacterial infection, suspected of having a bacterial infection, or at risk of having a bacterial infection, or with a complicated condition caused by bacterial colonization. In some respects, the subject has a skin and soft tissue infection (SSTI) or is at risk of having a skin and soft tissue infection (SSTI). In some respects, the subject has atopic dermatitis, folliculitis, pemphigus, or bullous pemphigoid or is at risk of having atopic dermatitis, folliculitis, pemphigus, or bullous pemphigoid. The compositions disclosed herein can inhibit the growth and / or activity of bacteria. The bacteria may be any bacteria possessing the agrC signaling system. In some respects, the bacteria are Staphylococcus. In some respects, the bacteria are Staphylococcus aureus or Staphylococcus epidermidis. In some respects, Staphylococcus aureus is methicillin-resistant Staphylococcus aureus (MRSA), methicillin-sensitive Staphylococcus aureus (MSSA), vancomycin-intermediate Staphylococcus aureus (VISA), or vancomycin-resistant Staphylococcus aureus (VRSA). In other respects, Staphylococcus aureus is Staphylococcus aureus LAC strain, Staphylococcus aureus USA 100 strain, Staphylococcus aureus MW2 strain, or Staphylococcus aureus MN EV strain.

[0067] In some respects, Ent-testosterone can be formulated into pharmaceutical compositions. These compositions can be used in various dosage forms to suit preferred routes of administration. Therefore, 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, transdermal, rectal, etc.). The compositions can be applied to mucosal surfaces, for example, via sprays or aerosols to the nasal or respiratory mucosa. The compositions can also be administered via sustained-release or delayed-release methods, and / or washed off or eluted from medical dressings (e.g., bandages).

[0068] In specific terms, the pharmaceutical composition may be in any suitable form, including but not limited to solutions, suspensions, emulsions, sprays, aerosols, or any mixtures. For example, the formulation may be in conventional topical dosage forms, such as creams, ointments, aerosol formulations, non-aerosol sprays, gels, lotions, etc. The composition may also contain one or more additives, such as adjuvants, skin penetration enhancers, colorants, fragrances, flavoring agents, humectants, thickeners, etc.

[0069] In some aspects, the compositions used in this method may comprise pharmaceutically acceptable carriers, excipients, and / or stabilizers, which may be in the form of lyophilized formulations or aqueous solutions. In some aspects, the acceptable carriers, excipients, and / or stabilizers are non-toxic to the subject at the doses and concentrations used and may include buffers (such as phosphates, citrates, and other organic acids); antioxidants (such as ascorbic acid and methionine); preservatives (such as octadecyl dimethyl benzyl ammonium chloride, hexamethyl ammonium chloride, benzalkonium chloride, benzyl chloride, phenol, butanol or benzyl alcohol, alkyl p-hydroxybenzoates such as methyl or propyl p-hydroxybenzoates, catechol, resorcinol, cyclohexanol, 3-pentanol; m-cresol); low molecular weight (less than about 10 residues) peptides; proteins, Examples of active ingredients include 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 ions; metal complexes (such as zinc-protein complexes); and / or nonionic surfactants such as Tween, Pluronics, or polyethylene glycol (PEG).

[0070] In some aspects, the compositions described herein may comprise liposomes loaded with ENT-testosterone. In some aspects, the liposomes used in this invention can be prepared by reverse-phase evaporation, wherein the lipid components comprise phosphatidylcholine, cholesterol, and polyethylene glycol-modified phosphatidylethanolamine (PEG-PE). In some aspects, the liposomes of this invention can be extruded through a filter membrane with a specified pore size to obtain liposomes of the desired diameter.

[0071] In some respects, ENT-testosterone can be encapsulated in microcapsules, which can be prepared, for example, by coagulation technology or interfacial polymerization, such as hydroxymethyl cellulose or gelatin microcapsules and polymethyl methacrylate microcapsules, for use in colloidal drug delivery systems (such as liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or crude emulsions.

[0072] In some respects, the compositions described herein may be sterile. This can be easily achieved, for example, by filtration through a sterile filter membrane. In some respects, the compositions of the present invention may be placed in a container with a sterile access port, such as an intravenous infusion bag or vial with a stopper that can be punctured by a hypodermic needle.

[0073] In some respects, the compositions described herein can be formulated into unit-dose dosage forms, such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral, rectal, or inhalation / blowing administration. The formulations can be presented in unit-dose form and prepared using methods known in the pharmaceutical field. Methods for preparing compositions comprising a pharmaceutically acceptable carrier include the step of binding ENT-testosterone to a carrier constituting one or more excipients. Typically, formulations can be prepared by homogeneously and / or tightly binding the active compound with a liquid carrier, a finely pulverized solid carrier, or both, followed by shaping the product into the desired dosage form if necessary.

[0074] In some respects, the emulsion mixtures described herein can be prepared by mixing Ent-testosterone with Intralipid™ or its components (soybean oil, egg yolk phospholipids, glycerol, and water).

[0075] Suitable emulsions can be prepared using commercially available fat emulsions (such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™, and / or Lipiphysan™). The active ingredient can be dissolved in a premixed emulsion composition or in an oil (such as soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil, or almond oil), and then mixed with phospholipids (such as egg yolk phospholipids, soybean phospholipids, or soybean lecithin) and water to form an emulsion. It should be understood that other ingredients, such as glycerol or glucose, can be added to adjust the osmotic pressure of the emulsion. Suitable emulsions typically contain up to about 20% oil, for example, from about 5% to about 20%. The fat emulsion may contain fat droplets with a particle size of about 0.1 μm to about 1.0 μm (especially about 0.1 μm to 0.5 μm) and a pH range of about 5.5 to 8.0.

[0076] In some aspects, the compositions herein for inhalation or intranasal administration may include solutions and suspensions of pharmaceutically acceptable aqueous solvents, or organic solvents, or mixtures thereof, as well as powders. In some aspects, the liquid or solid compositions herein may contain suitable pharmaceutically acceptable excipients as described above. In other aspects, the compositions may be administered orally or via nasal inhalation to produce local or systemic effects.

[0077] In some aspects, the composition may be in a sterile, pharmaceutically acceptable solvent and nebulized. In some aspects, the nebulized solution herein may be inhaled directly from a nebulizer, or the nebulizer may be connected to a face mask, oxygen tent, or intermittent positive pressure ventilator. In some embodiments, the solution, suspension, or powder composition may be administered via a suitable delivery device, preferably orally or nasally.

[0078] In some aspects, the compositions disclosed herein may comprise a buffer system. As used herein, a "buffer system" is a composition consisting of one or more buffers, wherein a "buffer" refers to a compound used to resist pH changes upon dilution or addition of an acid / base. Buffers include, but are not limited to, potassium metaphosphate, potassium phosphate, sodium acetate, anhydrous and sodium citrate dihydrate, and other substances known to those skilled in the art. In some aspects, any pharmaceutically acceptable organic or inorganic buffer may be used. In some aspects, the total content of one or more buffers in the pharmaceutical compositions disclosed herein may be 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% by weight of the composition. In some aspects, the total content of one or more buffers in the pharmaceutical compositions disclosed herein may be from about 5% to 99%, about 10%, about 95%, or about 15% to 90% by weight of the composition.

[0079] In some aspects, the amount of one or more buffers may depend on the desired pH level of the composition. In some aspects, the pharmaceutical compositions disclosed herein may have a pH value of about 6 to about 9. In some aspects, the pharmaceutical compositions disclosed herein may have a pH value greater than about 8, greater than about 7.5, greater than about 7, greater than about 6.5, or greater than about 6.

[0080] In some aspects, the compositions disclosed herein may contain one or more preservatives. As used herein, "preservative" means an agent or combination of agents capable of inhibiting, reducing, or eliminating bacterial growth in a pharmaceutical preparation. Non-limiting examples of preservatives include methylparaben, propylparaben, isopropanol, and combinations thereof. In some aspects, any pharmaceutically acceptable preservative may be used. In some aspects, the total content of one or more preservatives in the pharmaceutical compositions disclosed herein may be 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% by weight of the composition. In some aspects, the total content of one or more preservatives in the pharmaceutical compositions disclosed herein may be from 5% to 99%, about 10%, about 95%, or about 15% to 90% by weight of the composition.

[0081] In some aspects, the compositions disclosed herein may comprise one or more surfactants. In some aspects, the surfactants may be synthetic, natural, or semi-synthetic. In some aspects, the compositions disclosed herein may comprise anionic surfactants, cationic surfactants, amphoteric surfactants, amphoteric surfactants, amphoteric electrolyte surfactants, nonionic surfactants having a steroidal skeleton, or combinations thereof. In some aspects, the total content of one or more surfactants in the compositions disclosed herein may be 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% by weight of the composition. In some aspects, the total content of one or more surfactants in the compositions disclosed herein may be from about 5% to about 99%, about 10%, about 95%, or from about 15% to about 90% by weight of the composition.

[0082] In some aspects, the compositions disclosed herein may comprise one or more stabilizers. As used herein, "stabilizer" means a compound used to stabilize an active ingredient and prevent it from undergoing physical, chemical, or biochemical processes that would otherwise reduce the therapeutic activity of the active ingredient. Suitable stabilizers include, but are not limited to: succinic anhydride, albumin, sialic acid, creatinine, glycine and other amino acids, nicotinamide, sodium acetyltryptophan, zinc oxide, sucrose, glucose, lactose, sorbitol, mannitol, glycerol, polyethylene glycol, sodium caprylate, and sodium saccharin, as well as other substances known to those skilled in the art. In some aspects, the total content of one or more stabilizers in the compositions disclosed herein may be 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% by weight of the composition. In some aspects, the total content of one or more stabilizers in the compositions disclosed herein may be from 5% to 99%, about 10%, about 95%, or about 15% to 90% by weight of the composition.

[0083] In some aspects, the compositions disclosed herein may comprise one or more osmotic pressure regulators. As used herein, "osmotic pressure regulator" refers to a compound that can be used to adjust the osmotic pressure of a liquid formulation. Suitable osmotic pressure regulators include, but are not limited to, glycerol, lactose, mannitol, glucose, sodium chloride, sodium sulfate, sorbitol, trehalose, and other substances known to those skilled in the art. The osmotic pressure in the composition may be expressed in milliosmol / L (mOsm / L) and may be determined by methods known in the art. In some aspects, the osmotic pressure of the compositions disclosed herein is calculated using the vapor pressure reduction method. In some respects, the amount of one or more osmotic modifiers contained in the pharmaceutical compositions disclosed herein can enable the composition to achieve an osmotic pressure 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 respects, the osmotic pressure of the compositions described herein may be 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 aspects, the osmotic pressure of the pharmaceutical compositions described herein can be from about 100 mOsm / L to about 1000 mOsm / L, from about 200 mOsm / L to about 800 mOsm / L, from about 250 mOsm / L to about 500 mOsm / L, from about 250 mOsm / L to about 350 mOsm / L, from about 250 mOsm / L to about 320 mOsm / L, or from about 280 mOsm / L to about 320 mOsm / L. In some aspects, the total content of one or more osmotic pressure regulators in the pharmaceutical compositions disclosed herein, based on the total weight of the composition, can be 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%. In some respects, the total content of one or more osmotic pressure regulators in the pharmaceutical compositions disclosed herein may be from about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% by weight of the composition.

[0084] In some aspects, the concentrations of Ent-testosterone disclosed herein may be predetermined or standard concentrations. In some aspects, the concentrations of Ent-testosterone disclosed herein may be from about 1 microgram / mL (mg / ml) to about 500 mg / ml, from about 1 mg / ml to about 250 mg / ml, from about 1 mg / ml to about 200 mg / ml, from about 1 mg / ml to about 150 mg / ml, from about 1 mg / ml to about 100 mg / ml, from about 1 mg / ml to about 75 mg / ml, from about 1 mg / ml to about 50 mg / ml, from about 0.1 mg / ml to about 100 mg / ml, or other suitable concentrations. In some aspects, Ent-testosterone is in lyophilized form. In some aspects, Ent-testosterone is diluted to a suitable concentration in a suitable solution prior to administration.

[0085] In some aspects, the composition may be provided in any suitable form, including but not limited to solutions, suspensions, emulsions, sprays, aerosols, or mixtures of any blended forms. In some aspects, Ent-testosterone is formulated as a topical application formulation. For example, the formulation may be applied as a cream, ointment, aerosol formulation, non-aerosol spray, gel, emulsion, etc. The composition may also contain one or more additives, such as adjuvants, skin penetration enhancers, colorants, fragrances, flavoring agents, humectants, thickeners, etc. In some aspects, the compositions disclosed herein comprise Ent-testosterone and petrolatum.

[0086] In some aspects, the compositions disclosed herein may be incorporated into a patch applied to a skin area to be treated. As used herein, a "patch" comprises at least a topical composition containing Ent-testosterone and a covering layer, such that the patch can be placed on the skin area to be treated. The patch may be designed to maximize drug delivery through the stratum corneum into the epidermis or dermis, shorten residence time, promote uniform absorption, and / or reduce mechanical detachment. In some aspects, when the intended use includes the treatment of skin infections, the patch is designed to minimize drug absorption into the circulatory system. Preferably, the patch components have a viscoelasticity similar to that of skin and conform to the skin during limb movement to prevent excessive shearing and peeling. Compared to conventional methods of administration, patches containing topical formulations offer the following advantages: (i) dose control by the patch surface area; (ii) constant rate of administration; (iii) longer duration of action (adhering to the skin for 1, 3, 7 days or longer); (iv) improved patient compliance; (v) non-invasive administration; and (vi) reversible action (i.e., the patch can be easily removed).

[0087] In one aspect, a patch suitable for this disclosure comprises at least: (1) a backing layer; and (2) a carrier in which the compound of the present invention is formulated. Patch systems suitable for implementing the present invention include, but are not limited to: matrix-type patches, reservoir-type patches, multi-laminate drug-in-adhesive-type patches, and monolithic drug-in-adhesive-type patches. A matrix patch comprises: a matrix containing the composition disclosed herein, an adhesive backing film overlay, and a preferred but not essential anti-adhesive liner. In some cases, an impermeable layer may be required to minimize drug migration to the backing film. In one aspect, a matrix containing the compound of the present invention is adhered to the skin via 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 anti-stick liner includes, but is not limited to, airtight, opaque or transparent polyester films with a thin coating of pressure-sensitive anti-stick liner (such as silicone-fluorosilicone, perfluorocarbon-based polymers).

[0088] In some respects, the dosage of Ent-testosterone can vary due to a variety of factors, including but not limited to: the specific microorganism to be treated, the subject's weight, physical condition and / or age, and the route of administration. Therefore, the absolute weight of Ent-testosterone contained in a particular unit dosage form can vary considerably, depending on the type of Ent-testosterone used, the type of infectious microorganism, the subject's age, weight and physical condition, and / or the route of administration. Those skilled in the art, after appropriately considering the above factors, can readily determine the appropriate dosage.

[0089] In some aspects, the compositions disclosed herein can deliver a sufficient amount of Ent-testosterone to a subject by administration, for example, in a dose range of about 100 ng / kg to 50 mg / kg. In some aspects, the compositions can provide a subject with an Ent-testosterone dose of about 10 μg / kg to about 5 mg / kg, for example, an Ent-testosterone dose of about 100 μg / kg to about 1 mg / kg.

[0090] In some aspects, the compositions described herein may further comprise one or more active agents or therapeutic agents. Alternatively, ENT-testosterone may be administered in combination with one or more other therapeutic agents for patients in need. Non-limiting examples of additional active agents 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: lincosamides (such as lincomycin or clindamycin), penicillins (such as nafcillin), cephalosporins (such as cefuroxime, ceftazidime (alone or in combination with avibactam), or cefoloza (alone or in combination with tazobactam)), glycopeptides (such as vancomycin, orivancin, dapavancin), lipopeptides (such as dapoxetine), aminoglycosides (such as gentamicin), oxazolidinones (such as linezolid, tedizolid, posizolid, or cycloserine), or tetracyclines (such as doxycycline). Exemplary antimicrobial therapeutics include: immunotherapeutic agents, such as antimicrobial antibody therapy and / or antimicrobial cytokine therapy.

[0091] In some respects, Ent-testosterone can be administered before or after a subject develops symptoms or clinical signs of bacterial infection. "Symptoms" refers to any subjective evidence of any disease or patient condition. "Signs" or "clinical signs" refers to objective physical examination findings relevant to a specific condition that can be detected by someone other than the patient. Treatment initiated before a subject develops symptoms or clinical signs of infection can be considered prophylactic treatment for a subject at "risk" of bacterial infection. As used herein, the term "at risk" means that the subject may actually have or not have the stated risk. For example, a subject at risk of an infectious condition is one who is in an area where individuals with that infectious condition have been identified, and / or may be exposed to a source of infection, even if the subject has not yet shown detectable signs of bacterial infection, and regardless of whether they carry subclinical levels of bacteria.

[0092] Therefore, administration of this composition can be performed before, during, or after the subject's first appearance of symptoms or clinical signs of the condition, or before, during, or after the subject's first exposure to the bacteria. Initiating treatment before the subject's first appearance of symptoms or clinical signs related to the condition may reduce the likelihood of developing clinical symptoms of the condition, alleviate the severity of symptoms and / or clinical signs, and may completely cure the condition compared to subjects not treated with this composition. Treatment initiated after the subject's first appearance of symptoms or clinical signs related to the condition can be considered therapeutic treatment for the subject, and may alleviate the severity of symptoms and / or clinical signs, and / or completely cure the disease compared to subjects not treated with this composition.

[0093] In some respects, administration of a composition containing Ent-testosterone can block, inhibit, or reduce tissue damage in a subject. In some respects, the tissue damage may be skin or other soft tissue injury. In some respects, administration of the composition can reduce tissue damage in a subject by at least 10%, such as 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher, compared to a subject before administration of the composition (baseline) or a treatment-naïve control subject. In some respects, administration of a composition containing the disclosed Ent-testosterone can reduce tissue damage in a subject by at least about 20%. Tissue damage can be determined by any method known in the art for examining apoptosis or cell viability, including bioluminescence imaging or flow cytometry.

[0094] IV. Treatment Methods This disclosure further provides a method for treating a subject suffering from a bacterial infection or a complicated condition caused by bacterial colonization.

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

[0096] In a further aspect, this disclosure covers a method of treating a subject suffering from or at risk of a bacterial skin and soft tissue infection (SSTI), the method comprising administering to the subject a composition comprising Ent-testosterone. In some aspects, SSTI is an abscess, cellulitis, folliculitis, MRSA (methicillin-resistant Staphylococcus aureus) infection, staphylococcal infection (Staphylococcus aureus or Staphylococcus epidermidis), erysipelas, impetigo, boil, or carbuncle. The bacteria may be Staphylococcus spp. In some aspects, the bacteria are Staphylococcus aureus. In some aspects, the Staphylococcus aureus is methicillin-resistant Staphylococcus aureus (MRSA), methicillin-sensitive Staphylococcus aureus (MSSA), vancomycin-intermediate Staphylococcus aureus (VISA), or vancomycin-resistant Staphylococcus aureus (VRSA). In some respects, Staphylococcus aureus is Staphylococcus aureus LAC strain, Staphylococcus aureus USA 100 strain, Staphylococcus aureus MW2 strain, or Staphylococcus aureus MN EV strain.

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

[0098] In some aspects, the methods disclosed herein include treating subjects who have or are at risk of Staphylococcus aureus infection. The method includes administering to the subject a composition containing Ent-testosterone. The Staphylococcus aureus may be methicillin-resistant Staphylococcus aureus (MRSA), methicillin-sensitive Staphylococcus aureus (MSSA), vancomycin-intermediate Staphylococcus aureus (VISA), or vancomycin-resistant Staphylococcus aureus (VRSA). In some aspects, the Staphylococcus aureus is Staphylococcus aureus LAC strain, Staphylococcus aureus USA 100 strain, Staphylococcus aureus MW2 strain, or Staphylococcus aureus MN EV strain.

[0099] In another aspect, the method of the present invention includes treating a subject suffering from or at risk of Staphylococcus epidermidis infection. The method involves administering a composition containing Ent-testosterone to the subject.

[0100] In another aspect, the method includes limiting tissue damage in a subject caused by bacterial virulence-associated proteins by administering a composition containing Ent-testosterone to the subject. In these aspects, the tissue is skin tissue or other soft tissue. The bacteria may be Staphylococcus bacteria. In some aspects, the bacteria are Staphylococcus aureus or Staphylococcus epidermidis. In some aspects, the Staphylococcus aureus is methicillin-resistant Staphylococcus aureus (MRSA), methicillin-sensitive Staphylococcus aureus (MSSA), vancomycin-intermediate Staphylococcus aureus (VISA), or vancomycin-resistant Staphylococcus aureus (VRSA). In some aspects, the Staphylococcus aureus is Staphylococcus aureus LAC strain, Staphylococcus aureus USA 100 strain, Staphylococcus aureus MW2 strain, or Staphylococcus aureus MN EV strain.

[0101] This disclosure also provides a method for reducing the virulence of Staphylococcus spp., comprising contacting Staphylococcus spp. bacteria with a composition containing Ent-testosterone. In some aspects, the Staphylococcus spp. bacteria are Staphylococcus aureus or Staphylococcus epidermidis. In some aspects, the Staphylococcus aureus is methicillin-resistant Staphylococcus aureus (MRSA), methicillin-sensitive Staphylococcus aureus (MSSA), vancomycin-intermediate Staphylococcus aureus (VISA), or vancomycin-resistant Staphylococcus aureus (VRSA). In some aspects, the Staphylococcus aureus is Staphylococcus aureus LAC strain, Staphylococcus aureus USA 100 strain, Staphylococcus aureus MW2 strain, or Staphylococcus aureus strain MN EV.

[0102] In some aspects of this method, the disclosed bacteria can be any bacteria with quorum sensing capabilities. Any bacteria possessing the agrC signaling system can be treated using the methods disclosed herein, such as Pseudomonas aeruginosa.

[0103] Subjects may be any individual at risk of bacterial infection or diagnosed with a bacterial infection, such as staphylococcal infection. Staphylococcal infections (especially Staphylococcus aureus) can occur in many parts of the body, including skin infections and conditions such as osteomyelitis, endocarditis, pyogenic arthritis, pneumonia, abscess formation, gastroenteritis, scalded skin syndrome, and toxic shock syndrome. Staphylococcus aureus strains are considered the leading cause of nosocomial infections in the United States. Staphylococcal infections (especially Staphylococcus aureus) caused by antibiotic-resistant strains are often serious and life-threatening. High-risk groups for staphylococcal infection include, but are not limited to: subjects with diabetes, cancer, vascular disease, eczema, or lung disease; subjects with burns; and subjects who have been in close contact with individuals infected with staphylococci. Further, those at higher risk for staphylococcal infection include: subjects in intensive care units, subjects who have undergone surgery, subjects undergoing dialysis, subjects using catheters / breathing tubes / feeding tubes, subjects with implanted medical devices (such as pacemakers, artificial joints, or heart valves), and subjects with weakened immune systems (such as those diagnosed with HIV infection, subjects receiving organ / tissue transplant anti-rejection therapy, and subjects receiving chemotherapy). Subjects may include children and infants in kindergartens, preschools, or schools. Further, subjects may be individuals at risk of or diagnosed with Staphylococcus epidermidis infection. Staphylococcus epidermidis infection can cause wound infections, boils, sinusitis, endocarditis, and other inflammations.

[0104] The methods disclosed herein may comprise a composition containing an effective amount of Ent-testosterone. This effective amount may be a therapeutically effective amount. The composition may be administered to a subject in a dose-dependent manner. In some aspects of this method, suitable and non-limiting examples of the dosage according to this disclosure may be from about 1 ng / kg to about 5000 mg / kg. However, generally, the dosage range typically used for adult treatment is: from about 0.0001 mg / kg / day to about 0.0010 mg / kg / day, from about 0.0010 mg / kg / day to about 0.010 mg / kg / day, from about 0.010 mg / kg / day to about 0.10 mg / kg / day, from about 0.10 mg / kg / day to about 1 mg / kg / day, from about 1 mg / kg / day to about 200 mg / kg / day, and from about 200 mg / kg / day to about 5000 mg / kg / day. For example, the dosage can be from about 1 mg / kg / day to about 100 mg / kg / day, such as about 2-10 mg / kg / day, about 10-50 mg / kg / day or about 50-100 mg / kg / day. The dosage may also be selected from approximately 1 mg / kg, approximately 5 mg / kg, approximately 10 mg / kg, approximately 15 mg / kg, approximately 20 mg / kg, approximately 25 mg / kg, approximately 30 mg / kg, approximately 35 mg / kg, approximately 40 mg / kg, approximately 45 mg / kg, approximately 50 mg / kg, approximately 60 mg / kg, approximately 70 mg / kg, approximately 80 mg / kg, approximately 90 mg / kg, approximately 100 mg / kg, approximately 125 mg / kg, approximately 150 mg / kg, approximately 175 mg / kg, approximately 200 mg / kg, approximately 250 mg / kg, approximately 300 mg / kg, approximately 400 mg / kg, approximately 500 mg / kg, approximately 600 mg / kg, approximately 700 mg / kg, approximately 800 mg / kg, approximately 900 mg / kg, approximately 1000 mg / kg, approximately 1100 mg / kg, approximately 1200 mg / kg, approximately 1300 mg / kg, etc. 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 subjects once daily or multiple times daily. In some aspects, the disclosed Ent-testosterone may be administered every 2, 3, 4, 5, 6, 7, 14, or 30 days. The disclosed Ent-testosterone may be administered over time periods of approximately 1 day to approximately 1 year, approximately 1 day to approximately 1 week, approximately 3 days to approximately 1 month, approximately 2 weeks to approximately 6 months, or approximately 2 months to approximately 4 months. The disclosed Ent-testosterone may also be administered over time periods of approximately 1 day, approximately 7 days, approximately 30 days, approximately 60 days, approximately 120 days, or approximately 180 days and beyond. In some aspects, the disclosed Ent-testosterone may be administered over time periods of approximately 57 weeks, approximately 148 weeks, approximately 208 weeks, or indefinitely.

[0106] The dosage and dosing interval can be adjusted individually to provide an adequate amount to maintain therapeutic or preventative effects. For example, depending on the route of administration, specific indications, and the prescribing physician's judgment, Ent-testosterone can be administered once weekly, several times a week (e.g., every other day), once daily, or multiple times daily. In cases of local administration or selective absorption (e.g., topical application), the effective local concentration of Ent-testosterone may be independent of plasma concentrations. Those skilled in the art can optimize the effective dosage without excessive experimentation.

[0107] In some embodiments, the method of administration of Ent-testosterone reduces tissue damage in the subject. In some aspects, tissue damage is skin or other soft tissue injury. In some aspects, the method of administration of Ent-testosterone reduces tissue damage in the subject by at least 10%, such as 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher, compared to tissue damage in the subject before administration of Ent-testosterone (baseline) or in untreated control subjects. In some aspects, the method of administration of Ent-testosterone reduces tissue damage in the subject by at least about 20%. Tissue damage can be determined by any method known in the art for examining apoptosis or cell viability, including bioluminescence imaging or flow cytometry.

[0108] In one respect, the administration method of Ent-testosterone reduces staphylococcal-induced hemolysis in subjects. In some respects, the administration method of Ent-testosterone reduces staphylococcal-induced hemolysis in subjects by at least 10%, such as 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher, compared to staphylococcal-induced hemolysis in subjects before Ent-testosterone administration (baseline) or in untreated control subjects. In some respects, the administration method of Ent-testosterone reduces staphylococcal-induced hemolysis in subjects by at least about 5%.

[0109] In another aspect, the administration method of Ent-testosterone can reduce staphylococcal-induced neutrophil killing in subjects. In some aspects, this administration method of Ent-testosterone can reduce staphylococcal-induced neutrophil killing in subjects (baseline) before Ent-testosterone administration or in untreated control subjects by at least 10%, such as 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher. In some aspects, the administration method of Ent-testosterone can reduce staphylococcal-induced neutrophil killing in subjects by at least about 5%.

[0110] In a further aspect, the disclosed Ent-testosterone administration method can reduce one or more adverse reactions in subjects due to bacterial infection (e.g., Staphylococcus aureus), or alleviate the subject's disease state. Non-limiting examples of adverse reactions include hemolysis, toxic shock, biofilm formation, and toxicity. In some aspects, the Ent-testosterone administration method can reduce hemolysis, toxic shock, biofilm formation, and toxicity in subjects by at least 10%, such as 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher, compared to hemolysis, toxic shock, biofilm formation, and toxicity in subjects before Ent-testosterone administration (baseline) or in untreated control subjects.

[0111] The methods disclosed herein can be administered simultaneously, 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 therapy regimen should take into account the compatibility of the desired therapeutic agents and / or procedures and the expected therapeutic effect. It should also be understood that the therapies used can produce the desired effect for the same condition (e.g., ENT-testosterone can be used concurrently with other anti-inflammatory agents, immunomodulators, antibiotics, analgesics, or anticancer agents) or achieve different effects (e.g., control of any adverse reactions).

[0112] In some aspects, the disclosed methods may further include adjustments to the single or multiple administration of Ent-testosterone as described herein. For example, adjustments to one or more steps of the administration method may include modifications or changes to one or more features or aspects of one or more steps in the disclosed methods. For example, in one aspect, the method may be modified by changing the amount of Ent-testosterone administered to the subject, changing the frequency of administration of Ent-testosterone to the subject, or changing the duration of administration of one or more of the disclosed Ent-testosterones to the subject.

[0113] V. Reagent Kit This disclosure provides kits containing Ent-testosterone for use in conjunction with the methods of this disclosure. The kit may contain a composition containing Ent-testosterone, and instructions for administering Ent-testosterone to a subject in need. The kit may also contain other therapeutic agents, such as antibiotics that can be administered in combination with Ent-testosterone. In some aspects, the kit may further contain sterile, pharmaceutically acceptable carriers, buffers, or other diluents. Kits provided herein typically include instructions for performing the method. Instructions in the kit may be attached to packaging material or as a package insert. While instructions are typically in written or printed form, they are not limited to this. This disclosure covers any medium capable of storing and communicating such instructions to the end user, including but not limited to electronic storage media (such as disks, magnetic tapes, cassettes, chips), optical media (such as CD-ROMs), etc. As used herein, the term "instructions" may include an Internet address providing the instructions.

[0114] The kit disclosed in this invention may have a single container holding the disclosed Ent-testosterone, which may or may not contain any other components; alternatively, the kit may have separate containers for each desired reagent. When providing combination therapy, the components may be pre-mixed into a single solution in a molar equivalent ratio, or with one component in excess of another. Alternatively, the Ent-testosterone and other therapeutic components in the kit may be stored separately in individual containers before administration to a patient.

[0115] When the reagent kit components are provided in the form of one or more liquid solutions, the liquid solution is preferably an aqueous solution, with sterile aqueous solutions being particularly preferred. However, the reagent kit components may also be provided as dry powders. When reagents or components are provided in dry powder form, they can be reconstituted by adding a suitable solvent. This solvent may also be placed in other containers.

[0116] The kit's containers typically include at least one vial, tube, flask, bottle, syringe, or other container for holding the disclosed Ent-testosterone and any other desired formulations, preferably appropriately aliquoted. In cases containing separate components, the kit typically also includes a second vial or other container for holding these components, enabling the administration of separate designed doses. The kit may also include a second / third container for holding sterile, pharmaceutically acceptable buffers or other diluents.

[0117] The kit may also include an apparatus for administering the disclosed Ent-testosterone to animals or patients, such as one or more needles, syringes, or even droppers, pipettes, or similar devices, for injecting the formulation into the animal or applying it to a diseased site on the body. The kits disclosed herein typically also include an apparatus for tightly housing vials and other components for commercial sale, such as injection-molded or blow-molded plastic containers in which the required vials and other devices can be placed and held.

[0118] Having described several aspects, those skilled in the art will understand that various modifications, alternative structures, and equivalent solutions can be adopted without departing from the spirit of the invention. Furthermore, to avoid unnecessarily obscuring this disclosure, certain well-known processes and elements have not been described. Therefore, this specification should not be considered a limitation of this disclosure.

[0119] Those skilled in the art will understand that the aspects disclosed herein are taught by way of example and not by way of limitation. Therefore, the content contained herein or illustrated in the accompanying drawings should be interpreted as illustrative and not restrictive. The appended claims are intended to cover all general and specific features described herein, as well as all statements relating to the scope of methods and components, which may fall within their scope for the sake of language.

[0120] Example The following embodiments are intended to illustrate several aspects of this disclosure. Those skilled in the art should understand that the techniques disclosed in the following embodiments are techniques that the inventors have found applicable and effective in the practice of this disclosure, and therefore can be considered preferred methods of its implementation. However, those skilled in the art should understand that many modifications can be made to the specific aspects disclosed without departing from the spirit and scope of this disclosure, still achieving the same or similar effects.

[0121] Materials and Methods mice Six- to nine-week-old male and female C57BL6 / J mice were purchased from Jackson Laboratory. C57BL6 / J wild-type, K14Cre+ / -(60) and Hsd3b6fl / fl Mice were bred and raised in a specific pathogen-free (SPF) barrier environment at the University of Texas Southwestern Medical Center in Dallas. Hsd3b6ΔSkin ( K14Cre+ / -; Hsd3b6fl / fl The establishment of the strain is detailed below. In all experiments, 3-5 mice were housed in each cage. All mice were placed in a 12-hour light:12-hour dark environment. Mice had free access to food and water, following a protocol approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Texas Southwestern Medical Center.

[0122] Using CRISPR / Cas9 genome editing technology, guide RNAs were designed to target the Hsd3b6 locus region, and a [missing information - likely a gene editing system] was constructed. Hsd3b6 The first coding exon of the gene is surrounded by loxPHsd3b6fl / fl (C57BL6 / J) mice at the locus (Fig. 745 S2A). Guide RNA was injected into fertilized C57BL / 6J embryos at the Mouse Genome Engineering Facility of the Children's Research Institute at the University of Texas Southwestern Medical Center. Healthy blastocysts were transferred into pseudopregnant mice. Offspring were screened by genome sequencing for testing. loxP The insertion site was determined, and the mice were then bred to homozygosity and backcrossed with wild-type C57BL / 6 mice. To obtain... K14Cre+ / -;Hsd3b6fl / fl(Hsd3b6ΔSkin) Mice, Hsd3b6fl / fl mice and K14Cre+ / - Mouse hybridization K14Cre+ / -;Hsd3b6fl / + Mice, then K14Cre; Hsd3b6fl / + mice and Hsd3b6fl / fl Mouse hybridization to obtain experimental group mice K14Cre; Hsd3b6fl / fl (Hsd3b6ΔSkin) and the corresponding control group Hsd3b6fl / fl The Hsd3b6fl / fl and Hsd3b6ΔSkin states were identified using PCR primers (Table 1) and 3% agarose gel electrophoresis.

[0123] Table 1: Staphylococcus aureus strains

[0124] Skin FACS analysis Wild-type infants aged 8-12 weeks, HSD3B6fl / flEar skin from HSD3B6Δskin mice was digested in RPMI medium with the following substances: 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 tissue was minced and incubated at 37°C and 1400 rpm for 1 hour using a thermal cycler, followed by filtration through a 70 μm cell separation filter. After washing, the cell pellet was resuspended in FACS buffer (PBS containing 3% BSA and 2 mM EDTA). The cell suspension was transferred to V-bottom 96-well plates (Corning Inc: Costar; 3894) for further processing. Cell viability was assessed using Ghost-Dye-Red-710 (Cytek; SKU 13-0871-T100). To prevent non-specific antibody binding, Fc receptor blocking staining was performed beforehand using anti-mouse CD16 / 32 antibody (BD Biosciences; catalog number 553142). Different cell populations were evaluated using the following antibodies: antibodies were resuspended in FACS buffer and incubated at 4°C for 15 minutes. These antibodies were: 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 the cell population was as follows: For total white blood cells, single viable cells were screened by gating the CD45+ marker, from which all T cells were identified as CD45+CD3+ cells, and macrophages as CD45+CD11b+F4 / 80+ cells. Cell collection was performed using a NovoCyte flow cytometer, and data analysis was conducted using NovoExpress software.

[0125] Immunofluorescence microscopy Mouse skin samples were formalin-fixed and paraffin-embedded at the UT Southwestern Histology Core facility. After dewaxing in xylene, samples were rehydrated with graded concentrations of ethanol. Heat-induced antigen retrieval was performed in 10 mM sodium citrate buffer. Sections were briefly washed and then blocked for 1 hour in blocking / permeabilization buffer (PBS + 5% goat serum + 0.5% Triton X-100). Sections were then 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), slides were incubated with their 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). The slides were then briefly washed with PBST and mounted with mounting medium containing 4',6-diamidinyl-2-phenylindole (DAPI) (SouthernBiotech; 0100-20). Images were acquired and processed using a ZEISS 780 confocal microscope.

[0126] Bacterial strains and plasmids Staphylococcus aureus strains (Table 1) were streaked onto tryptone soy agar (TSA) plates and incubated overnight at 37°C. Single colonies were selected and placed in TSB, incubated overnight at 150 RPM in a shaking incubator at 37°C, and then passaged at a 1:100 ratio to obtain bacteria in the mid-logarithmic growth phase. For fusion reporter strains, in vitro cultures were performed using TSB medium containing 10 μg / mL chloramphenicol. After centrifugation, the bacteria were washed and resuspended in TSB (in vitro experiments) or PBS (in vivo experiments). The HG003, ΔagrC, ΔagrBD, and ΔagrA mutant strains were provided by Dr. Ferric Fang. Staphylococcus aureus strains from atopic dermatitis skin were provided by Dr. Julie Segre and Dr. Heidi Kong. The remaining strains were from the strain collections of the Horswill and Harris-Tryon laboratories.

[0127] Construction of Staphylococcus aureus strains expressing the lux gene Integrated luxCDABEGThe gene cassette was transduced via phage 11 into Staphylococcus aureus strains HG003, ΔagrBD, and ΔagrC obtained from Ferric Fang's laboratory, producing AH6222 ( lux+ ), AH6224 ( lux+ ) and AH6223 ( lux+ ) strain.

[0128] In vitro chemiluminescence detection Staphylococcus aureus strains HG003, ΔagrBD, and ΔagrC express Lux ( 11::LL29 luxCDABEG The quorum sensing lux (pAmiAgrP3lux) plasmids (HG003(AH6225)agr type I, USA100(AH430) type II, and MW2(AH1747)agr type III) (52, 65, 66) were cultured in antibiotic-supplemented TSB and then transfected into fresh TSB containing steroid hormones at a ratio of 1:200. Experiments were performed in opaque sidewall, 96-well, clear-bottomed, tissue culture-treated plates with a final well volume of 200 μL. Bioluminescent signals (photons / 0.1 sec acquisition time) were detected using a BioTek H1 Synergy microplate reader. All experiments were performed in triplicate, with agri-type specific AIPs: AIP-Ⅰ (Peptide Institute, catalog number 4515-v), AIP-Ⅱ (Peptide Institute, catalog number 4516-v), and AIP-Ⅲ (Peptide Institute, catalog number 4517-v) serving as positive controls.

[0129] hemolysis test The hemolysis assay was performed as previously described, with the following modifications: Overnight cultures of HG003, ΔagrBD, and ΔagrC strains were inoculated at a ratio of 1:200 into 10 mL of TSB medium containing testosterone, AIP-I, or either solvent alone, at a concentration of 10 nM. Cells were allowed to grow to mid-log phase (OD 600 nm 0.6). One mL of culture supernatant was then separated using a Millex microscope with a pore size of 0.22 μm. ®The supernatant was sterilized by filtration using a sterile syringe filter (product number: SLGV033RS). The supernatant was diluted 1:1 with PBS, and 25 μL of human blood was added to each V-bottom 96-well plate. The plate was incubated at 37°C with shaking for 1 hour. After centrifugation at 1000 RPM for 10 minutes, the supernatant was transferred to a flat-bottom 96-well plate. Hemoglobin absorbance was measured at 541 nm using a BioTek H1 Synergy microplate reader. The percentage of hemolysis was calculated using the following formula: (A541 value of RBC-treated sample - A541 value of buffer) / (A541 value of water - A541 value of buffer). Buffer (PBS) = baseline value, water = 100% hemolysis.

[0130] Neutrophil killing assay Staphylococcus aureus-induced neutrophil killing was determined according to the previously described method. HG003, ΔagrBD, and ΔagrC strains were treated with 10 nM testosterone, AIP-I, or solvent, and cultured to mid-log (OD 600 nm 0.6). Purified human neutrophils (IQ Biosciences) were cultured at 1 × 10⁶ cells per well. 5 Cells were seeded at a density of 100 μL in 96-well plates containing 90 μL of RPMI medium. 10 μL of bacterial supernatant (final concentration 10%) was added. After incubation at 37°C and 5% CO2 for 3 hours, the cells were centrifuged at 250 g for 10 minutes, and the supernatant was collected. Lactate dehydrogenase (LDH) released from damaged cells was measured using an LDH cytotoxicity assay kit (Invitrogen, catalog number 2570393) as a marker of neutrophil lysis. Neutrophils cultured in 10% RPMI were used as a 0% lysis control, and neutrophils cultured in 0.2% Triton X-100 were used as a 100% lysis control; the percentage of neutrophil lysis was calculated.

[0131] Quantitative Real-Time PCR HG003, USA100 (AH3684), MW2 (AH843), ΔagrBD, ΔagrA, ΔagrC, and atopic dermatitis strains were treated with 10 nM testosterone and / or corresponding AIPs and cultured to mid-log (OD600nm 0.6). The bacterial pellet was collected and lysed at room temperature using lysis matrix B tubes containing 0.1 mm silica microspheres (MP lysis matrix tube, catalog number 174701) and lysin (Sigma, catalog number L7386). RNA was then purified using the RNeasy Mini Kit (Qiagen, catalog number 74104). RNA was quantified by absorbance at 260 nm, and purity was assessed using the 260 / 280 nm absorbance ratio. cDNA was synthesized using the RNA as a template using a high-capacity reverse transcription kit (Applied Biosystems, catalog number 01071619). Quantitative real-time PCR was performed using Power SYBR Green Master Mix (Applied Biosystems, catalog number 2749999) and the QuantStudio 7Flex real-time PCR system (Applied Biosystems) to amplify cDNA. Relative expression levels were calculated by comparing Ct values ​​(ΔΔCt method), and transcript abundance was normalized based on gyrA transcript abundance. Primer sequences are shown in Table 2.

[0132] Table 2: Primers for Quantitative RT-PCR and Gene Sequencing

[0133] RNA sequencing In summary, strain HG003 was cultured in TSB medium containing 10 nM testosterone, pregnenolone, or DMSO alone, with three replicates per group, until the absorbance at OD600 nm reached 0.6. Cells were collected and treated with RNA ProtectBacteria reagent (Qiagen, catalog number 76526). Cells were lysed with lysostaphin (Sigma, catalog number L7386), and RNA was purified using the RNeasy mini kit (Qiagen, catalog number 74104). Sample quality was confirmed using a bioanalyzer (Agilent). Ribosomal RNA (rRNA) was removed using the RiboCop Bacterial META Removal Kit (Lexogen). cDNA libraries were constructed at the University of Michigan Microbiomecore using the Lexogen CORALL RNA-seq Library Preparation Kit. Samples were barcoded, mixed, and sequenced at 125 × 125 paired ends using an Illumina HiSeq 2000 sequencer. QiagenCLC Genomics Workbench (version 21.0.5) was used with default settings (mismatch penalty: 2; insertion and deletion penalty: 3; length and similarity score: 0.8) to align and annotate raw sequencing reads in FASTQ format to the Staphylococcus aureus NCTC8325 reference genome (annotated sRNA(70)). CLC was used to normalize and calculate differential expression of uniquely aligned bacterial transcripts. All transcripts with FDR-corrected p-values ​​<0.05 were considered significantly different.

[0134] Staphylococcal skin infection Prior to the mouse infection study, mice were acclimatized to an animal biosafety level 2 (ABSL-2) facility. The study used age-, strain-, and sex-matched male / female C57BL / 6 mice. Hsd3b6fl / fl and Hsd3b6ΔSkin Mice. A previously described mouse model of Staphylococcus epidermidis exposure was established. In short: the skin on the back of anesthetized mice (2% isoflurane) was shaved and depilated (using nifedipine cream). After 24 hours, bioluminescent Staphylococcus aureus strains were cultured to mid-logarithmic phase, centrifuged to collect bacterial cells, and resuspended in PBS to prepare a solution containing 1×10⁻⁶ cells / mL. 6 CFU inoculum. Take a solution containing 1×10 6100 μL of CFU in PBS solution (with or without 10 nmol 900 testosterone, AIP-I, or an equal volume of solvent) was placed on sterile gauze and applied to the shaved skin area with a transparent biological occlusive dressing (Tegaderm; 3M, Henry Schein Medicals, catalog number 1622W), and fixed with a band-aid (BAND-AID, Johnson & Johnson, American White Cross, catalog number 1275033) for 4 days. Photons emitted by the luminescent bacteria were acquired using an IVISLumina3 imager and Living Image software (Xenogen, Alameda, CA) in automatic exposure mode. Bioluminescence image data were presented as pseudo-color scales (blue representing the lowest intensity signal, red representing the highest intensity signal) and superimposed on grayscale images. Using the image analysis tools in Living Image software, a circular analysis window covering a uniform area was used to measure the corresponding bioluminescence values ​​(total flux) and plot the post-infection day change curve. Mice were randomly assigned to the treatment group, and at the end of the experiment, the mice were euthanized humanely by carbon dioxide inhalation.

[0135] PSMα expression assay The relative expression levels of transcripts of the AgrA-dependent virulence factor phenol-soluble regulatory protein α (PSMα) were quantified using RT-PCR and compared with the expression levels of the bacterial housekeeping gene DNA gyrase (gyrA). Mean ± standard error was plotted.

[0136] In vivo experiments female HSD3B6 Δ皮肤 Mice were infected with the bioluminescent MRSA strain MRSA-lux via epidermal infection. The in vivo infection process was tracked over time using an in vivo imaging system (IVIS) capable of detecting bacterial bioluminescence at both two-dimensional and three-dimensional levels.

[0137] Quantitative and statistical analysis Data are expressed as mean ± standard error of mean. Although formal randomization was not used, mice were randomly assigned to experimental groups, and samples were processed in any order. Skin samples determined to be in the hair growth phase were excluded. Except for bioluminescence imaging data analyzed as described above, all statistical analyses were performed using GraphPad Prism software. Two-tailed Student's t-tests were used to assess the statistical significance of differences between the two treatment groups; one-way ANOVA was used to assess the statistical significance of differences between more than two treatment groups. Outliers were identified and removed using the Grubb test. RNA sequencing experiments were performed using CLC software to analyze expression data; all transcripts with a p-value <0.05 after FDR correction were considered statistically significant.

[0138] Example 1. Ent-testosterone reduced the expression of virulence genes in various Staphylococcus aureus strains. To evaluate the effectiveness 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 solvent, respectively. The relative expression of the AgrA-dependent virulence factor phenol-soluble regulatory protein α (PSMα) was quantified by RT-PCR and compared with the expression of the bacterial housekeeping gene DNA gyrase (gyrA). Figure 2 Ent-testosterone was found to inhibit the expression of PSMα in all strains of Staphylococcus aureus types I, II, III, and IV, while testosterone upregulated the expression of the virulence factor PSMα.

[0139] Example 2: Ent-testosterone reduces quorum sensing in mice The efficacy of Ent-testosterone was further validated through in vivo experiments. HSD3B6 Δ皮肤 Female mice were percutaneously infected with bioluminescent MRSA agr-P3 The lux reporter strain was treated with 10 nM Ent-testosterone, 10 nM testosterone, or a solvent, respectively. Compared with control mice, Hsd3b6 Δ皮肤 Mice secrete less testosterone through their skin. An in vivo imaging system (IVIS) capable of detecting bacterial bioluminescence at both two-dimensional and three-dimensional levels was used to track changes in the in vivo infection process over time. Figure 3 Representative images are shown at 1 hour, 2 hours, 3 hours, and 4 hours after processing. Figure 4 The average bioluminescence intensity per hour over 8 hours after treatment was plotted. Figure 3 and Figure 4 As shown, testosterone enhanced bioluminescence over time, while Ent-testosterone inhibited it. These results suggest that Ent-testosterone treatment can suppress agr-mediated bioluminescence, thus implying that Ent-testosterone can inhibit quorum sensing.

[0140] Example 3: Ent-testosterone dosage In addition, MRSA is used agr-P3 The lux reporter strain was tested in vitro with different doses of Ent-testosterone. MRSA agr- P3 The lux strain was treated with 10 nM, 100 nM, 1 µM, and 10 µM concentrations of Ent-testosterone, testosterone, AIPII, or solvent, and the changes in its bioluminescence over time were as follows: Figure 5 and Figure 6 As shown, Ent-testosterone reduced bioluminescence at all tested doses, indicating that Ent-testosterone inhibits MRSA.

[0141] Example 4: Testosterone enantiomers inhibit the pathogenicity of Staphylococcus aureus To investigate the specificity of the interaction between testosterone and the agr system, this study tested the effects of the stereoisomer of testosterone, the enantiomer-testosterone (ent-T), on the pathogenicity of Staphylococcus aureus. Similar to other non-natural enantiomers, ent-T possesses the same physicochemical properties as testosterone, but its six chiral centers are all mirror-image, leading to a change in the direction of polarized light rotation. Figure 7A Observations revealed that enantiomers possess unique receptor binding and signal transduction properties. This differs from other tested hormones that exhibit a neutral effect on the agr system. Figures 9A-9D Ent-testosterone can act as a broad-spectrum inhibitor of the agr quorum sensing system. Figure 2 , Figures 7B-7D , Figures 8A-8B Ent-testosterone can reduce Staphylococcus aureus-dependent hemolysis and neutrophil killing in type I agr strains. Figures 7B-7C Furthermore, Ent-testosterone can inhibit the expression of agr transcriptional readout in Staphylococcus aureus type II, III, and atopic dermatitis strains. Figures 7B-7D , Figure 8A Furthermore, topical application of Ent-testosterone to the skin of Staphylococcus aureus-infected mice can shut down quorum sensing in vivo. Figure 3 , Figure 4 , Figure 8B Therefore, ent-T, as a broad-spectrum inhibitor of Staphylococcus aureus quorum sensing, can act on different agr types, similar to known bacterial agr regulators, such as non-homologous AIPs (AIPs). Figures 7B-7C ).

[0142] Example 5: Ent-testosterone-mediated quorum sensing quenching The effect of ENT-testosterone on an infection model was investigated by disrupting the mouse skin barrier using a tape-peeling method. An established percutaneous skin infection protocol was employed. In short, mice were anesthetized with isoflurane before undergoing tape-peeling treatment (transcutaneous water loss TEWL = 20 g / m²). 2 / h), 10 after 24 hours 7 CFU of MRSA SAP 430 suspension (100µl) was applied to the back skin and spread evenly with a cotton swab. Mice were returned to their cages after the inoculum had dried for 10 minutes. Treatment was administered once daily, and Tegaderm dressings were not used during the treatment period. In the MRSA in vivo infection mouse model, no difference was observed between the control solvent treatment group and the ENT treatment group. However, a significant difference was observed between the TEWL treatment group and the control solvent group. Figures 10A-10B This may be because Ent-testosterone mediates quorum sensing quenching at the site of infection in the body. It inhibits the production of AIP, a member of the family of signaling molecules used in quorum sensing. Therefore, the proposed Ent-testosterone alleviates atopic dermatitis (AD)-related symptoms such as itching, redness, swelling, pain, and / or skin barrier disruption by inhibiting quorum sensing, thereby promoting skin barrier repair without eliminating Staphylococcus aureus, which is a component of the normal skin microbiome.

[0143] Example 6: Ent-testosterone inhibits quorum sensing in vivo Male mice were administered testosterone percutaneously at doses of 10 and 100 nanomolars. Immediately afterwards, the AH2659agrP3 reporter strain was administered, and agr kinetics were recorded hourly. A previously described Staphylococcus aureus epidermal exposure mouse model was used. Briefly, the dorsal skin of anesthetized mice (using 2% isoflurane) was shaved and depilated (using Nair cream). Twenty-four hours later, the bioluminescent Staphylococcus aureus strain was cultured to mid-logarithmic growth phase, and the cells were collected by centrifugation and resuspended in PBS to prepare a solution containing 1 × 10⁻⁶ cells. 6 CFU inoculum. Take a solution containing 1×10 6 100 μL of CFU in PBS was placed on sterile gauze, secured to the shaved skin area with a transparent biological occlusive dressing (Tegaderm; 3M, Henry Schein Medicals, catalog number 1622), and reinforced with a Band-Aid (BAND-AID, Johnson & Johnson, American White Cross, catalog number 1275033). Photons emitted by the luminescent bacteria were acquired using an IVISLumina3 imager and Living Image software (Xenogen, Alameda, California) in automatic exposure mode.

[0144] like Figures 11A-11BAs shown, ENT-testosterone has been found to suppress quorum sensing. No changes in body weight were observed among the treatment groups on the day of infection. Figure 11C There was no difference in TEWL measured among the treatment groups. Figure 11D ).

[0145] Furthermore, to verify whether high-dose ENT-testosterone could compete with testosterone on the skin, the experiment used incrementally increasing concentrations of ENT-testosterone in combination with testosterone, while simultaneously measuring the agrP3 luminescence value. Figure 12 As shown, in the presence of testosterone, it has been found that increased ENT-testosterone concentration reduces the luminescence of HG003 agrP3 in a dose-dependent manner.

[0146] Summary of Implementation Examples In summary, Ent-testosterone inhibits the expression of virulence factors and quorum sensing both in vitro and in vivo. These results suggest that Ent-testosterone can suppress bacterial virulence and therefore has therapeutic applications in treating complicated conditions caused by bacterial infections or colonization.

[0147] Unlike progesterone and estradiol, which have neutral effects on Staphylococcus aureus, the stereoisomer of testosterone, ENT-testosterone, reduces quorum sensing and decreases bacterial-induced hemolysis and neutrophil killing. ENT-testosterone also inhibits quorum sensing during Staphylococcus aureus infection. These findings suggest that Staphylococcus aureus responds to testosterone in an enantioselective manner: testosterone generates an activating signal, while its chiral enantiomer, ENT-testosterone, inhibits the same phenotype. Since natural hormones and their enantiomers have shown identical 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 androgens on bacterial membranes.

Claims

1. A composition for treating bacterial infections in subjects in need, the composition comprising an enantiomer of testosterone (Ent-testosterone).

2. A method for treating a subject suffering from or at risk of bacterial infection, the method comprising: The subject was administered a composition containing Ent-testosterone.

3. A method for treating a subject suffering from or at risk of bacterial skin and soft tissue infection (SSTI), the method comprising: The subject was administered a composition containing Ent-testosterone.

4. A method for treating a subject suffering from or at risk of suffering from complicated conditions caused by bacterial colonization, the method comprising: The subject was administered a composition containing Ent-testosterone.

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 Staphylococcus bacteria.

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 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 LAC strain, Staphylococcus aureus USA 100 strain, Staphylococcus aureus MW2 strain and / or Staphylococcus aureus MNEV strain.

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 being.

12. The composition or method according to any one of claims 1-11, wherein, compared with an untreated control subject, Ent-testosterone reduces the expression of virulence factor or virulence-related protein genes by at least 10%; (b) reduces staphylococcal-induced hemolysis by at least 10%; and / or (c) reduces staphylococcal-induced neutrophil killing by at least 5%.

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

14. The composition or method according to claim 12, wherein the virulence-related protein gene is agrII type.

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

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

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

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

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

20. The composition or method according to any one of claims 1-19, 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 tissues of a subject caused by bacterial virulence-associated proteins, the method comprising: The subject was administered a composition containing Ent-testosterone.

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

24. A method for reducing the virulence of Staphylococcus spp. bacteria, the method comprising: The Staphylococcus bacteria are brought into contact with a composition containing an effective amount of Ent-testosterone.

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

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

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

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