TUBERCULOSIS THERAPEUTIC COMPOUNDS AND METHODS

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims benefit of U.S. Provisional Patent Application Serial No. 62/421 ,629, filed November 14, 2016, herein incorporated by reference in its entirety.

GRANT STATEMENT

This invention was made with government support under Grant Number TR001 1 1 1 awarded by the National Institutes of Health. The government has certain rights in the invention.

TECHNICAL FIELD

The subject disclosure is directed to tuberculosis therapeutic compounds and methods. Provided are therapeutic approaches for treating tuberculosis infection in the lungs of patients, as well as extrapulmonary therapeutic effects.

BACKGROUND

With 1 .5 million deaths annually from tuberculosis (TB) and increasing numbers of drug resistant TB cases, including for example multiple drug resistant (MDR) TB and extensively drug resistant (XDR) TB, there is a need for new TB therapies [1 ]. Along with new drugs effective against resistant Mycobacterium tuberculosis {Mtb) strains, strategies and therapeutic approaches to shorten the duration of therapy (6 months for drug sensitive TB, greater than 2 years for drug resistant TB) are needed to simplify treatment, improve adherence, limit periods of transmission and improve therapeutic outcomes.

Existing therapeutic approaches and compounds for treating TB are inadequate. Advances in TB treatment, including novel compounds and therapeutic approaches, are provided herein to address the current deficiencies in existing treatments. SUMMARY

This summary lists several embodiments of the presently disclosed subject matter, and in many cases lists variations and permutations of these embodiments. This summary is merely exemplary of the numerous and varied embodiments. Mention of one or more representative features of a given embodiment is likewise exemplary. Such an embodiment can typically exist with or without the feature(s) mentioned; likewise, those features can be applied to other embodiments of the presently disclosed subject matter, whether listed in this summary or not. To avoid excessive repetition, this Summary does not list or suggest all possible combinations of such features.

In some embodiments, provided herein are methods of treating tuberculosis (TB) in a subject, the methods comprising providing a subject in need of treatment, providing an inhalable formulation of pyrazinoic acid (POA), and administering an effective amount of the inhalable formulation of POA to the subject by inhalation, wherein the TB in the subject is treated. In some embodiments, the inhalable formulation of POA comprises a dry powder of POA alone or in salt form. In some embodiments, the inhalable formulation of POA further comprises an aerodynamic particle size distribution (APSD) in a range suitable for pulmonary delivery. In some embodiments, the inhalable formulation of POA further comprises an APSD of about 1 μΓΠ to about 5 μηι. In some embodiments, the effective amount of the inhalable formulation of POA is less than an amount of POA or other TB therapeutic compound given orally. In some embodiments, the effective amount of the inhalable formulation of POA is about 2 mg/kg to about 5 mg/kg.

In some embodiments, administering the inhalable formulation of POA is more effective than treating TB with an enhaled pyrazinoic acid ester (PAE), and in some embodiments as effective as a higher does of orally administered pyrazinamide (PZA). In some embodiments, the subject is a human subject has TB or a related condition. In some embodiments, the TB in the subject comprises multiple drug resistant (MDR) TB or extensively drug resistant (XDR) TB. In some embodiments, treating the TB comprises eliminating or substantially reducing a bacterial infection comprising Mycobacterium tuberculosis {Mtb) or related bacterial strains.

In some embodiments, treating the TB further comprises a systemic therapeutic effect or host-directed effect comprising a resolution of granulomas in the spleen or liver, and/or eliminating or reducing Mtb in spleen. In some embodiments, treating the TB has a preferential effect on primary granulomatous lesions over secondary granulomatous lesions in the lung. In some embodiments, the inhalable formulation of POA is administered as an adjunctive therapy to orally administered rifampicin (RIF).

Also provided herein are methods of inducing antibacterial and host- directed effects against a Mycobacterium tuberculosis {Mtb) infection in a subject, the methods comprising providing a subject suffering from a Mtb infection, providing an inhalable formulation of pyrazinoic acid (POA), and administering an effective amount of the inhalable formulation of POA to the subject by inhalation, wherein the inhaled POA has an antibacterial effect on the Mtb infection in lungs and induces a host-directed effect against granulomas in the spleen and liver. In some embodiments, the antibacterial effect on the Mtb infection comprises substantially reducing or clearing the Mtb as determined by a sputum assay. In some embodiments, the host- directed effect against granulomas in the spleen and liver comprising a resolution of granulomas, as shown by their smaller size in the spleen or liver.

In some embodiments, the inhalable formulation of POA comprises a dry powder of POA alone or in salt form. In some embodiments, the inhalable formulation of POA further comprises an aerodynamic particle size distribution (APSD) in a range suitable for pulmonary delivery. In some embodiments, the inhalable formulation of POA further comprises an APSD of about 1 -5μΓη . In some embodiments, the effective amount of the inhalable formulation of POA is about 2 mg/kg to about 5 mg/kg.

In some embodiments, the TB comprises multiple drug resistant (MDR) TB and/or extensively drug resistant (XDR) TB. In some embodiments, the methods further comprise administering the inhalable POA, in combination with other therapies, to treat primary and secondary granulomatous lesions.

Provided herein are therapeutic compositions, comprising an aerosolized form of pyrazinoic acid (POA) with an aerodynamic particle size distribution (APSD) in a range suitable for pulmonary delivery, and an excipient or carrier selected from the group consisting of sugars, peptides, lipids and buffers. In some embodiments, the therapeutic composition is configured to treat tuberculosis (TB) when administered by inhalation. In some embodiments, an aerosolized formulation of POA comprises a spray dried powder of POA alone or in salt form. In some embodiments, the aerosolized formulation of POA comprises an APSD of about 1 -5μΓη . In some embodiments, the sugars comprise one or more of maltodextrin, mannitol, trehalose, lactose, and/or dextrose. In some embodiments, the peptides/polypeptides comprise one or more of leucine, lysine, glycine, polyleucine (dileucine, trileucine), and/or polylysine (dilysine). In some embodiments, the lipids comprise one or more of lecithin (phosphatidyl choline), phosphatidylethanolamine and/or magnesium stearate. In some embodiments, the buffers comprise one or more of phosphate buffered saline and/or cholesterol.

Accordingly, it is an object of the presently disclosed subject matter to provide tuberculosis therapeutic compounds and methods. This and other objects are achieved in whole or in part by the presently disclosed subject matter. Further, an object of the presently disclosed subject matter having been stated above, other objects and advantages of the presently disclosed subject matter will become apparent to those skilled in the art after a study of the following description, Drawings and Examples.

BRIEF DESCRIPTION OF THE DRAWINGS

The presently disclosed subject matter can be better understood by referring to the following figures. The components in the figures are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the presently disclosed subject matter (often schematically). In the figures, like reference numerals designate corresponding parts throughout the different views. A further understanding of the presently disclosed subject matter can be obtained by reference to an embodiment set forth in the illustrations of the accompanying drawings. Although the illustrated embodiment is merely exemplary of systems for carrying out the presently disclosed subject matter, both the organization and method of operation of the presently disclosed subject matter, in general, together with further objectives and advantages thereof, may be more easily understood by reference to the drawings and the following description. The drawings are not intended to limit the scope of this presently disclosed subject matter, which is set forth with particularity in the claims as appended or as subsequently amended, but merely to clarify and exemplify the presently disclosed subject matter.

For a more complete understanding of the presently disclosed subject matter, reference is now made to the following drawings in which:

FIGS. 1A and 1 B are graphical summaries of data showing the Mtb burden in guinea pig lung (FIG. 1A) and spleen (FIG. 1 B) following four weeks of treatment across the following treatment groups: untreated (diamond), oral RIF (inverted triangle), oral RIF plus 1 -2 mg/kg inhaled POA dry powder (circle), oral RIF plus 1 -2 mg/kg inhaled liquid PAE1 (square), and oral RIF plus oral PZA 25 mg/kg (triangle); and

FIGS. 2A and 2B are scanning electron microscopy images of POA with additives (FIG. 2A), and POA leucine salt particles (FIG. 2B).

DETAILED DESCRIPTION

The presently disclosed subject matter now will be described more fully hereinafter, in which some, but not all embodiments of the presently disclosed subject matter are described. Indeed, the presently disclosed subject matter can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the presently disclosed subject matter.

While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

All technical and scientific terms used herein, unless otherwise defined below, are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to techniques employed herein are intended to refer to the techniques as commonly understood in the art, including variations on those techniques or substitutions of equivalent techniques that would be apparent to one of skill in the art. While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

In describing the presently disclosed subject matter, it will be understood that a number of techniques and steps are disclosed. Each of these has individual benefit and each can also be used in conjunction with one or more, or in some cases all, of the other disclosed techniques.

Accordingly, for the sake of clarity, this description will refrain from repeating every possible combination of the individual steps in an unnecessary fashion. Nevertheless, the specification and claims should be read with the understanding that such combinations are entirely within the scope of the invention and the claims.

Following long-standing patent law convention, the terms "a", "an", and "the" refer to "one or more" when used in this application, including the claims. Thus, for example, reference to "a cell" includes a plurality of such cells, and so forth.

Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.

As used herein, the term "about," when referring to a value or to an amount of a composition, dose, mass, weight, temperature, time, volume, concentration, percentage, etc., is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1 %, in some embodiments ±0.5%, and in some embodiments ±0.1 % from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.

The term "comprising", which is synonymous with "including" "containing" or "characterized by" is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. "Comprising" is a term of art used in claim language which means that the named elements are essential, but other elements can be added and still form a construct within the scope of the claim.

As used herein, the phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. When the phrase "consists of" appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.

As used herein, the phrase "consisting essentially of limits the scope of a claim to the specified materials or steps, plus those that do not materially affect the basic and novel characteristic(s) of the claimed subject matter.

With respect to the terms "comprising", "consisting of", and "consisting essentially of", where one of these three terms is used herein, the presently disclosed and claimed subject matter can include the use of either of the other two terms.

As used herein, the term "and/or" when used in the context of a listing of entities, refers to the entities being present singly or in combination. Thus, for example, the phrase "A, B, C, and/or D" includes A, B, C, and D individually, but also includes any and all combinations and subcombinations of A, B, C, and D.

Disclosed herein are methods, treatments, therapies and compositions for treating TB using inhaled pyrazinoic acid (POA). Inhaled POA can in some embodiments be effective on the largest category of pyrazinamide (PZA) resistant Mtb strains, and can in some aspects have the potential to improve the efficacy of current TB treatment regimens.

PZA, rifampicin (RIF), isoniazid (INH) and ethambutol (EMB) comprise current multi-drug therapies for TB and particularly drug sensitive TB [3]. When first added to the regimen, PZA shortened treatment time from 12 to 6 months, and PZA is the only drug in the regimen considered to be irreplaceable without a loss of efficacy [4, 5]. PZA is unique among TB drugs in eliciting sterilizing and synergistic effects in vivo when combined with other drugs, such as INH [6, 7] and RIF [8, 9]. PZA is also included in many MDR-TB treatment regimens [10] and often included when testing new TB drug candidates [1 1 ]. PZA is a prodrug that is converted to its active moiety POA [12]. However, the mechanism of anti-TB activity of PZA/POA has not previously been completely elucidated. At best, the mechanism of anti-TB activity of PZA/POA has previously been attributed to action on numerous targets, including fatty acid synthesis [13], trans-translation [14, 15], membrane potential and integrity [16], pantothenate biosynthesis [17, 18], and the host immune response [19-21 ].

Following inhalation, Mtb is phagocytosed by macrophages and replicates intracellular^. Ultimately, a TH1 immune response develops, leading to macrophage activation and the formation of granulomas that control Mtb. However, even in the face of a TH1 response, Mtb can persist long-term in granulomas in a latent state and later reactivate to cause TB [22, 23]. Most TB drugs only work on replicating Mtb and/or do not penetrate the poorly vascularized granuloma [24, 25]. In contrast, PZA has a unique activity profile that helps explain its sterilizing effect [26]. PZA is active in acidic pH and effective on hypoxic, nutrient starved and non- replicating Mtb [17, 27-29]. Macrophages and granulomas can be acidic and the granuloma is hypoxic and nutrient limiting [30, 31 ]. Further, when sequestered in granulomas a population of Mtb is in a state of non- replicating/slow replicating persistence [25, 32]. Additionally, PZA can penetrate granulomas. In TB patients receiving treatment, PZA is detected in both the necrotic acellular core and the surrounding cellular regions of granulomas [33].

Therefore, given the unique attributes of PZA, the rise in PZA resistance (about 50% of MDR strains are PZA resistant) is concerning [34- 36]. Most PZA resistant strains harbor mutations in the Mtb pncA gene [2, 37 and 38], which encodes the PncA pyrazinamidase responsible for the conversion of PZA to the active moiety POA. When added directly to Mtb, POA has anti-Mb activity [39-41 ]. Thus, treating TB directly with POA could be a potential strategy to overcome PZA resistance due to PncA-deficiency. However, it has long been thought, and clearly documented, that direct treatment with POA as the drug in the dosage form, herein referred to as the POA dosage form, is not feasible, albeit for unknown reasons. Indeed, when tested as a monotherapy for the ability to treat TB in mice, orally delivered POA was significantly less efficacious than oral PZA delivered at equivalent doses, and higher oral POA doses are likely not feasible without significant tolerability problems [43]. Moreover, when given orally, POA poorly diffuses into lung epithelial lining fluid (ELF) compared to PZA [43]. Thus, prior to the instant disclosure, the use of POA alone was considered an ineffective therapy or treatment for TB, MDR TB, XDR TB and related conditions.

Despite the contraindication for the use of the POA dosage form as a therapy for TB and related conditions, the instant disclosure is based, at least in part, on the surprising discovery that inhalation based POA therapeutic approaches can be unexpectedly effective in treating TB, MDR TB and/or XDR TB. Particularly, although not wishing to be bound by any particularly theory or understanding of the mechanism of action, the instant disclosure is based on the direct delivery of POA to the lungs by inhalation, which can in some embodiments achieve higher POA concentrations in the lung and ELF than oral administration, all the while maintaining low or relatively low systemic levels. Moreover, by using the pulmonary route of delivery, relatively low doses of POA, compared to those thought to be clinically effective, exhibit surprisingly high efficacy in treating TB and related conditions.

Thus, the instant disclosure provides at least the following innovative aspects: 1 ) it challenges assumptions of POA dosage forms as a TB therapeutic approach; 2) it provides POA delivered as a dry powder aerosol directly to the lung, the site of Mtb infection, rather than by the conventional route of oral delivery employed for PZA; 3) it provides a low dose of drug (such as about 2 to about 5mg/kg), below what is usually considered therapeutic, which is made possible with the pulmonary route of delivery; 4) it provides a systemic therapeutic effect following inhalation of low doses of drug, which is surprising but supported by the disclosed data; and 5) it provides, in some embodiments, POA in combination with RIF to exploit known synergistic interactions of RIF and PZA, which could, in some aspects, contribute to the efficacy of inhaled POA.

By directly delivering drugs, e.g. POA, to the site of Mtb infection in the lungs, inhaled therapies have the potential to rapidly clear the airway, to prevent transmission, and to supplement drug therapy by conventional routes to shorten treatment duration. By maximizing the drug concentration at infected sites, inhaled therapies as disclosed herein can also reduce drug dosage and related side effects, which can improve compliance. As an alternative to parenteral administration, this route also has the advantage of eliminating needles, medical waste and cold storage.

More particularly, in some embodiments of the presently disclosed subject matter, dosing with inhaled POA comprises a strategy to treat the largest category of PZA resistant Mtb, and when used as a supplement to current oral multi-drug therapy, it can in some embodiments shorten treatment times. Thus, as disclosed herein, POA delivery by inhalation can achieve locally high concentrations at the site of infection, but systemically low doses, which can in some aspects, be effective as an adjunctive therapy for TB and related conditions.

Thus, contrary to the long-held belief that the POA dosage form is generally an ineffective TB therapy, the instant disclosure is based, at least in part, on the discovery that POA is an important drug component for TB. This is based at least in part on studies where pyrazinoic acid esters (PAEs) were administered (again based on the assumption that POA alone would not work). In addition, dry powders prepared of a small proportion of propyl pyrazinoate (PAE 1 ; about 5% to about 15%) and larger proportion of POA (about 20% to about 60%), including for example a dry powder comprising about 30.5% POA, about 8.5% PAE 1 , about 30.5% maltodextrin and about 30.5% leucine, were more effective in treating TB in an animal model of disease than the ester (PAE) alone. Esters are subject to hydrolysis in the host organism by ubiquitous esterases or related enzymes. The data demonstrating efficacy of the POA ester alone, where hydrolysis is likely to occur, and its enhancement by the presence of POA dry powder formulation, also referred to as POA dry powder herein, suggests that POA alone is responsible for the effect. This is a unique observation of therapeutically effective delivery of POA as the primary drug in the dosage form. Moreover, the instant disclosure is based on the discovery, as disclosed herein, that aerosol delivery to the lungs can in some aspects be beneficial, since POA delivered orally is not as efficacious at conventional (high) doses, i.e. it is not conventional to deliver POA orally [43].

Systemic effects of the POA dry powder (seen in the spleen and liver), which are rarely seen with aerosols designed to treat TB, are observed at drug concentrations that are below the usual therapeutic dose of pyrazinamide or other oral administrations. Thus, an aspect of the instant disclosure is the benefit of providing a TB therapy at very low doses, particularly as compared to traditional oral TB therapeutics, that is effective by inhalation.

Moreover, the systemic effects also establish that the disclosed POA dry powder can, in some embodiments, be effective in treating extrapulmonary TB, as well as pulmonary TB. For example, as disclosed herein, the histopathology indicates that there can in some embodiments be a significant resolution of granulomas in the spleen and liver. This finding suggests a host-directed effect in addition to an antibiotic effect usually associated with POA and related compounds. Overall, inhaled POA was found to be effective against pulmonary infection as measured by reducing bacterial burden of CFU in lungs and by resolving lung granulomas. Additionally, the inhaled POA therapies disclosed herein have host-directed systemic effects as shown by effects of reducing bacteria in the spleen and resolving granulomas in the spleen and liver.

Thus, in some embodiments, methods of treating tuberculosis (TB) in a subject are provided. Such methods can comprise providing a subject in need of treatment, providing an inhalable formulation of POA, and administering an effective amount of the inhalable formulation of POA to the subject by inhalation, wherein the TB is treated. The inhalable formulation of POA can comprise a dry powder of POA alone or in salt form, and can have an aerodynamic particle size distribution (APSD) in a range suitable for pulmonary delivery, e.g. about 1 -5 μηι.

Notably, the effective amount of the inhalable formulation of POA is less than an amount of POA or other TB therapeutic compound given orally, e.g. about 2 mg/kg to about 5 mg/kg. Moreover, administering the inhalable formulation of POA can in some aspects be more effective than treating TB with a pyrazinoic acid ester (PAE), as demonstrated by a greater decrease in Mtb infection and/or reduced granulomas. Moreover, in some embodiments the inhalable formulation of POA can be more effective than treating TB with PZA, even when PZA is delivered at a higher dose. The TB to be treated can comprise multiple drug resistant (MDR) TB and/or extensively drug resistant (XDR) TB.

Treating TB can comprise eliminating or substantially reducing a bacterial infection comprising Mtb and related bacterial strains. Such a reduction in bacterial infection can be evidenced by a reduced bacterial burden colony-forming unit (CFU), and/or by resolving granulomas, particularly in the lungs. In some embodiments sputum assays can be conducted to determine clearance of these bacterial organisms in clinical disease, or early bactericidal assays in clinical trials. Moreover, histological data indicates that treating TB based on the instant disclosure can reduce lung granulomas (fewer number and smaller), which is directly relevant to the pulmonary disease and bacterial infection. Thus, also provided herein are treatments that eliminate or substantially reduce lung granulomas.

Additionally, in some aspects treating TB further comprises a systemic therapeutic effect, or host-directed effect, comprising a resolution of granulomas, as shown by fewer and smaller sized granulomas, including in the spleen and liver.

Also provided herein are methods of inducing antibacterial and host- directed effects, in vivo, against a Mtb infection in a subject. Such a method can comprise providing a subject suffering from a Mtb infection, providing an inhalable formulation of POA, and administering an effective amount of the inhalable formulation of POA to the subject by inhalation, wherein the inhaled POA has an antibacterial effect, in vivo, on the Mtb infection and induces a host-directed systemic effect, such as against granulomas in the spleen and liver, which is relevant to extrapulmonary disease.

As shown in the Examples and data herein, the disclosed compositions, i.e. inhalable POA powders, can in some aspects have a preferential effect on primary lesions, indicating a novel mechanism of efficacy. That is, based on the instant disclosure, inhaled POA dry powders can be provided as a therapy or treatment for primary granulomas that arise during initial infection, which is atypical for existing TB therapies that target secondary lesions, i.e. granulomas that arise later in infection as a result of hematogenous spread of bacteria. Thus, inhaled POA can, in some embodiments, be coupled with existing TB therapies to cooperatively target primary as well as secondary lesions. Moreover, given the pulmonary and extrapulmonary effects, inhaled POA can also be provided, alone or in conjunction with an existing TB therapeutic, to treat TB both at the source, i.e. lungs, as well as systemically, i.e. extrapulmonary. Thus, in some embodiments, provided herein are combined therapies and therapeutic approaches employing the disclosed POA compounds in conjunction with existing TB therapeutic compounds, to cooperatively treat TB in the lungs and systemically, including treatment of primary and secondary granulomatous lesions. Therapeutic compositions for treating TB and related conditions are provided. Such compositions can comprise an aerosolized form of POA with an aerodynamic particle size distribution (APSD) in a range suitable for pulmonary delivery and an excipient and/or carrier, including for example sugars, peptides/polypeptides, lipids and/or buffers.

The therapeutic compositions can be configured to TB when administered by inhalation. The aerosolized formulation of POA can comprise a spray dried powder of POA alone or in salt form, and can have an APSD in a range of about 1 -5μΓη.

The excipient and/or carriers can include, but are not limited to sugars including maltodextrin, mannitol, trehalose, lactose, and/or dextrose; peptides/polypeptides including leucine, lysine, glycine, polyleucine (dileucine, trileucine), and/or polylysine (dilysine); lipids including but not limited to lecithin (phosphatidyl choline), phosphatidylethanolamine and/or magnesium stearate; and/or buffers including but not limited phosphate buffered saline and/or cholesterol.

The subjects treated or to be treated in the disclosed methods, systems and approaches, or for which the disclosed compounds and compositions are designed, are desirably human subjetst, although it is to be understood that the principles of the disclosed subject matter indicate that the compositions and methods are effective with respect to invertebrate and to all vertebrate species, including mammals, which are intended to be included in the term "subject". Moreover, a mammal is understood to include any mammalian species in which screening is desirable, particularly agricultural and domestic mammalian species.

The disclosed methods and compounds are particularly useful in the treatment of warm-blooded vertebrates. Thus, the presently disclosed subject matter concerns mammals and birds.

More particularly, provided herein is the treatment of mammals such as humans, as well as those mammals of importance due to being endangered (such as Siberian tigers), of economical importance (animals raised on farms for consumption by humans) and/or social importance (animals kept as pets or in zoos) to humans, for instance, carnivores other than humans (such as cats and dogs), swine (pigs, hogs, and wild boars), ruminants (such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels), and horses. Also provided is the treatment of birds, including the treatment of those kinds of birds that are endangered, kept in zoos, as well as fowl, and more particularly domesticated fowl, i.e., poultry, such as turkeys, chickens, ducks, geese, guinea fowl, and the like, as they are also of economical importance to humans. Thus, provided herein is the treatment of livestock, including, but not limited to, domesticated swine (pigs and hogs), ruminants, horses, poultry, and the like.

In some embodiments, the subject to be used in accordance with the presently disclosed subject matter is a subject in need of treatment and/or diagnosis. In some embodiments, a subject can have or be believed to TB or related disease, condition or phenotype. EXAMPLES

The following examples are included to further illustrate various embodiments of the presently disclosed subject matter. However, those of ordinary skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the presently disclosed subject matter.

EXAMPLE 1

Testing inhaled PAEs and POA as TB therapy

Liquid and dry powder formulations of propyl pyrazinoate (PAE1 ) and POA suitable for generation of respirable aerosols were prepared. A liquid formulation was prepared for nebulization of PAE1 in ethanol/phosphate buffered saline as follows 15:25:60 v/v PAE:EtOH:PBS. To prepare the POA dry power formulation, spray drying was used (Buchi Model 290, Mini Spray drier) with 8.5% PAE1 , 30.5% pyrazinoic acid (POA), 30.5% maltodextrin and 30.5% leucine. Thus, the dry powder is composed of a small proportion of PAE 1 and larger proportion of POA and will be referred to as POA dry powder herein. Spray drying parameters were tested and the final dry powder formulation was assessed by HPLC and MS analysis. Custom-built nose-only exposure chambers were used for delivery of POA dry powder and liquid PAE1 . The powders were dispersed from a powder insufflation system and liquids dispersed from an Aeroneb (2.5-4.0μΓη volume median diameter, Aerogen) vibrating mesh nebulizer.

Next, the efficacy of inhalation therapy with POA dry powder formulation or nebulized liquid PAE1 for treating TB in guinea pigs was evaluated.

Guinea pigs were first infected with virulent Mtb for four weeks and then treated daily with drugs for an additional four weeks. Study groups received oral rifampicin (RIF) (a component of standard current multidrug TB therapy) with or without inhalable preparations of POA dry powder or nebulized liquid PAE1 using custom-built exposure chambers. One group of animals was treated with oral RIF plus the inhaled POA dry powder. A second group was treated with oral RIF plus the inhaled nebulized liquid formulation of PAE1 . Both inhaled formulations were delivered at a relatively low dose of 1 -2 mg/kg. There was also a group of animals that received combined oral RIF plus oral PZA at a higher dose of 25 mg/kg. Finally there were groups of Mtb infected animals that received only oral RIF or no therapy whatsoever. Following completion of the four week treatment period, animals were sacrificed and necropsy performed. Organ homogenates were plated on agar. After three weeks incubation, the number of bacteria (colony forming units; CFU) in homogenates of lung, spleen and lymph node was determined to measure the bacterial burden and evaluate the efficacy of treatment (FIGS. 1 A and 1 B).

FIGS. 1A and 1 B summarize data showing the Mtb burden in guinea pig lung (FIG. 1A) and spleen (FIG. 1 B) following four weeks of treatment across the following treatment groups: untreated (diamond), oral RIF (inverted triangle), oral RIF plus 1 -2 mg/kg inhaled POA dry powder (circle), oral RIF plus 1 -2 mg/kg inhaled liquid PAE1 (square), and oral RIF plus oral PZA 25 mg/kg (triangle). All RIF treatment was 50mg/kg. Bacterial burden was determined by plating organ homogenate on 7H10 agar. In FIG. 1A, lung burden was determined for all five groups of six animals/group. In FIG. 1 B, spleen burden was determined for all groups of six animals/group. Groups were compared using the non-parametric Kruskal-Wallis test followed by a Dunn's test to calculate multiplicity adjusted p values ( ^* p<0.01 ). The level of detection is indicated in the graph as a dotted line. Animals with no detectable CFU were counted as having CFU at the level of detection.

Evaluation of the bacterial burden in lungs (FIG. 1A) indicates that treatment with oral RIF alone (inverted triangles) did not significantly reduce the lung burden of Mtb compared to untreated animals. In contrast, combined therapy of oral RIF plus inhaled POA dry powder (circles) resulted in a significant reduction in CFU in lungs when compared to untreated animals. Interestingly, a statistically significant effect was not seen with oral RIF plus the inhaled liquid PAE1 formulation (squares). Thus, when combined with RIF, the dry powder formulation of POA appeared to perform better than the liquid PAE1 formulation in controlling the lung bacterial burden. Like oral RIF combined with the inhaled dry powder of POA, oral RIF combined with oral PZA (triangles) at 25mg/kg significantly reduced the lung burden compared to untreated animals.

These findings that inhaled POA dry powders were as effective as oral PZA at an approximately 10X higher dose reveals that relatively low doses of POA are effective when delivered by inhalation, which can in some embodiments have significant value for limiting treatment side-effects and compliance problems.

In the spleens (FIG. 1 B), in combination with oral RIF, both the inhaled POA dry powder (circles) and liquid PAE1 (squares) formulations produced a significant reduction in bacterial burden compared to untreated animals as did the oral PZA (triangles) dose of 25mg/kg. It is worth noting that there were multiple animals with undetectable CFU in these three treatment groups and again there was a trend with the inhaled POA dry powder having a more pronounced effect in reducing the CFU burden, in this case below the level of detection, than the inhaled liquid PAE1 formulation. Treatment with oral RIF alone (inverted triangles) did not lead to a significant reduction in spleen burden versus untreated animals. The effects in the spleen are particularly interesting in revealing systemic effects of inhaled doses of drug. The CFU burden in tracheobronchial lymph nodes was also evaluated. In this case, all treatment groups including the group receiving only oral RIF exhibited a significant reduction in bacterial burden in comparison to untreated animals and no significant differences were found between groups.

Tissues were also collected for histopathology as another means of assessing treatment efficacy. Routine histologic examination of the lung, tracheobronchial lymph node, spleen, liver, and kidney was performed on all 6 animals in each of the four treatment group by a board-certified veterinary pathologist. In considering all organs assessed across all groups, the animals treated with oral RIF plus inhaled POA dry powder had the least severe histologic changes, with the smallest and least well developed pulmonary granulomas overall and mild hepatic inflammation. In comparison to this group, animals treated with oral RIF plus either inhaled liquid PAE1 or oral PZA at a 10X higher dose (25mg/kg) had somewhat more severe pathologic changes, having a higher number and more progressed and necrotic pulmonary granulomas, and also more severe hepatic inflammation while the pathologic changes in animals treated only with RIF were the most severe, evident in the number and progression of pulmonary granulomas and severity of hepatic inflammation. Importantly, no damage to lung or tracheal tissues was observed with either of the inhaled POA dry powder or liquid PAE1 formulations tested. Further details regarding histological analysis can be found in Example 2 below.

Taken together, these data indicate that an inhaled POA dry powder formulation (about 20% to about 60%) is effective in controlling TB. Indeed, these data support the finding that powder or aerosolized POA delivered via inhalation can in some embodiments provide an effective TB therapy. Further, these results demonstrate that an inhaled dry powder formulation is efficacious because a dry powder is a stable formulation, with advantages of being inexpensive, easier to use, requiring no special storage conditions, electrical supply, or medical oversight beyond initial inhaler training. These studies represent an example of an inhaled TB drug product.

EXAMPLE 2

Evaluating TB therapies and their histopathological effects

Groups 1 -5 of guinea pigs were infected with Mtb for four weeks (wks) and then groups of animals were treated for an additional four weeks (see Table 1 ). Groups receiving oral rifampicin (RIF) were given a dose at 50mg/kg. At eight weeks post-infection guinea pigs were sacrificed for necropsy. Group 6 refers to animals that were infected with Mtb for four weeks and sacrificed prior to the initiation of any treatment.

Table 1.

A summary of examined tissue sections, by organ, is provided below, for each of lung, tracheobronchial lymph nodes, spleen and liver.

Lung- Histologic description:

Randomly scattered throughout the lung parenchyma were variably sized and progressed granulomas consisting of epithelioid macrophage with associated fibrin, fewer foam cells, surrounding lymphocytes, and rare heterophils (pseudoeosinophils). Some granulomas contained a central, variably developed necrotic center with abundant eosinophilic and cellular debris. Some necrotic cores contained abundant central mineralization.

Lung- Morphologic Diagnosis:

Moderate to severe pulmonary granulomas with mineralization (see interpretation below for specific differences amongst treatment groups) were observed.

Lung- Interpretation:

Pulmonary granulomas were present and staged (Stages 1 -4 in severity) as described in Turner et al. [80] with minor modifications to the staging scheme. When histologic parameters did not align with the measurement criteria, histologic features were used for staging rather than measurement. For a summary of pulmonary histologic findings, see Tables 2 and 3.

Treatment groups 1 (oral RIF plus inhaled POA dry powder) and 3 (oral RIF plus oral PZA (PZA given at 10x dose compared to POA)) had the lowest percent of lung tissue affected by granulomas of all the groups. Further, the percentage of granulomas across a group that progressed to Stage 4 varied between treatment groups, with Group 1 having the least proportion of Stage 4 granulomas (5-10%) of all the treatment groups. Stage 4 granulomas possess necrotic centers, often with mineralization. These features of Stage 4 granulomas are hallmarks of primary lesions that result from initial M. tuberculosis inhalation and they differ from secondary lesions that are non-necrotic and a result of hematogenous reseeding of the lung as infection progresses. The finding of Group 1 having the least Stage 4 granulomas and, correspondingly, the highest proportion of less progressed and non-necrotic granulomas indicates a preferential effect of inhaled POA dry powder on primary lesions. The Group 1 effect on primary lesions differs from the other treatment groups 2-4 and also from prior studies [83, 84 and 85] of oral therapies of M. tuberculosis infected guinea pigs in which treatments preferentially resolve secondary lesions. The effect of inhaled POA dry powder versus other treatments on different categories of granulomas indicates a novel mechanism of efficacy.

Thus, inhaled POA was found to be effective against pulmonary infection as measured by reducing bacterial burden of CFU in lungs and by resolving lung granulomas. Additionally, the inhaled POA therapies disclosed herein were found to have host-directed systemic effects, as shown by reduced bacteria in the spleen and resolving granulomas in the spleen and liver.

To elaborate, these data demonstrate that inhaled POA dry powders can be provided as a therapy or treatment for primary granulomas contain M. tuberculosis that arise during initial infection, which is atypical for existing TB therapies that preferentially target secondary lesions, i.e. granulomas that arise later in infection as a result of hematogenous spread of bacteria. Thus, inhaled POA can, in some embodiments, be coupled with existing TB therapies to cooperatively target primary as well as secondary lesions. Moreover, given the pulmonary and extrapulmonary effects, inhaled POA can also be provided, alone or in conjunction with an existing TB therapeutic, to treat TB both at the source, i.e. lungs, as well as systemically, i.e. extrapulmonary.

For (untreated) Group 5, the granulomas tended to encompass a slightly larger area of the surface area of the examined lung sections, and a mix of Stage 4 and less progressed granulomas was evident, as expected. Tracheobronchial lymph nodes- Histologic description:

In (untreated) Groups 5 and 6, the lymph node is enlarged 10-100X normal size, with normal architecture completely effaced and replaced by abundant macrophages, surrounded and infiltrated by epitheliod macrophages, foam cells, lymphocytes, plasma cells, heterophils (pseudoeosinophils), and surrounded by a fibrous capsule. Any remaining lymph node is minimal and completely peripheralized by the granuloma. Many nodes display central necrosis, with abundant cellular debris and sometimes mineralization. Histologic findings in the examined sections are similar for Groups 1 , 2, 3, and 4, although an occasional node in each group is less affected, either displaying early granuloma formation or having the morphology of a reactive node with enlarged, well developed lymphoid follicles and increased macrophages.

Tracheobronchial lymph nodes- Morphologic Diagnosis:

Tracheobronchial lymph nodes, Groups 1 , 2, 3, 4, 5: Granulomatous lymphadenitis, severe, multifocal to coalescing with caseous necrosis and mineralization.

Tracheobronchial lymph nodes, Groups 1 , 2, 3, 4: Hyperplastic

(reactive) node [occasional].

Tracheobronchial lymph nodes- Interpretation:

Consistent with experimental parameters of infection with M. tuberculosis, lymph nodes were effaced by granuloma formation, a classic manifestation of mycobacterial infection.

Spleen- Histologic description:

The white pulp of (8 wks untreated) Group 5 was expanded by large aggregates of macrophages centered on the lymphoid follicles. Macrophages had abundant cytoplasm (epithelioid macrophages) and rarely displayed multinucleation. The surrounding red pulp had increased circulating heterophils (pseudoeosinophils) and epithelioid macrophages. In contrast, the red pulp of (treated) Group 1 displayed increased numbers of circulating heterophils and macrophages with occasional small aggregates of macrophages, but did not display granuloma formation. Treatment groups 2, 3, and 4 displayed histologic changes within this spectrum, with increased circulating heterophils and macrophages and varying numbers of granulomas focused within lymphoid follicles.

Spleen- Morphologic Diagnosis:

Spleen, Group 1 : Increased circulating lymphocytes with occasional minimal granulomatous splenintis. Spleen, Group 2, 3, 4, 6: Granulomatous splenitis, moderate, multifocal to coalescing, with increased circulating lymphocytes.

Spleen, Group 5: Granulomatous splenitis, severe, multifocal to coalescing, with increased circulating lymphocytes.

Spleen- Interpretation:

Consistent with experimental infection with Mtb, the white pulp was variably expanded by aggregates of epithelioid macrophages. The exception to this was Group 1 , in which there were occasional small aggregates of macrophages but no overt granulomas, suggestive of resolution of prior splenic granulomas. These histologic findings were also consistent with reduced systemic spread to the spleen; however, this possibility seemed unlikely given that the animals were infected for 4 wks prior to initiation of treatment, and animals examined at the time of initiation of treatment displayed splenic granulomas. Groups 2, 3, 4, and 6 showed variable numbers of granulomas. Overall the number of splenic granulomas between the spleens were as follows: Group 1 <2<6<3,4<5.

Liver- Histologic description:

Across the groups there were two types of inflammatory lesions present, as follows:

(Groups 2, 4) Scattered, small granulomas comprised of aggregates of macrophages with abundant cytoplasm with smaller numbers of surrounding lymphocytes and associated fibrin. Group 4 displayed some areas of hepatocyte necrosis. Group 5 displayed larger granulomas with associated hepatocyte degeneration and necrosis. Group 1 displayed minimal inflammation.

Also, there was a variably mild to moderate number of lymphocytes with fewer macrophages and occasional plasma cells that centered on and expands from portal tracts.

Liver- Morphologic Diagnosis:

Liver, Group 1 : Mild portal reactive hepatitis. Liver, Group 2: Mild multifocal granulomatous hepatitis with moderate portal reactive hepatitis.

Liver, Group 3: Mild portal reactive hepatitis.

Liver, Group 4: Mild multifocal granulomatous hepatitis with mild portal reactive hepatitis.

Liver, Group 5: Moderate to severe, multifocal granulomatous hepatitis with moderate portal reactive hepatitis.

Liver, Group 6: Moderate multifocal granulomatous hepatitis with mild to moderate portal reactive hepatitis.

Liver- Interpretation:

Across the experimental groups examined, there were two types of hepatic inflammatory lesions present: granulomas and chronic periportal hepatitis. The variably sized, randomly distributed hepatic granulomas in groups 2, 4, 5, and 6 were a hallmark of extrapulmonary Mtb infection. Conversely, lymphoplasmacytic periportal hepatitis is an inflammatory infiltrate predominated by lymphocytes and plasma cells that tracks the vascular architecture of the liver and is a nonspecific, reactive inflammatory pattern secondary to chronic disease of the alimentary tract. Treatment Group 1 displayed mild, nonspecific reactive periportal hepatitis.

Table 2.

Table 3.

Summary of extrapulmonary histologic findings

Group 1 Group 2 Group 3 Group 4 Group 5 Group 6

TBLN granulomas severe severe severe severe severe severe

Spleen granulomas minimal moderate moderate moderate severe moderate

Liver granulomas none Mild none mild severe moderate reactive mild to mild moderate mild mild moderate

hepatitis moderate

When compared to all other groups of treated or untreated animals, Group 1 animals (treated with inhaled dry powder POA) exhibited the least histopathologic lesions across all organs examined. Group 1 animals had a lower percent of tissue comprised of granulomas (in lungs, spleen and liver) and Group 1 animals also had less severe granulomas in spleen, liver and lung according to the scoring scheme. Notably, Group 1 animals had the least Stage 4 granulomas of all treatment groups. Group 6 animals that were sacrificed at 4 weeks of Mtb infection prior to a treatment period had granulomas in the livers and worse pathology in spleen, liver and lung than the Group 1 animals, which indicates the POA dry powder treatment received by Group 1 animals was effective at treating existing disease pathology.

EXAMPLE 3 Formulating POA for inhaled POA therapies

Aerodynamic particle size distribution (APSD) in the range suitable for pulmonary delivery (about 1 -5 μηι) was determined using Aerodynamic Particle Size time-of-flight laser instruments (TSI) and Next Generation Impactors (Copley Scientific) [67]. POA was formulated as a leucine salt (POA-leu). A POA salt can have the combined advantage of increasing the solubility of POA and moderating the pH, which can help obviate concerns regarding local adverse effects of delivering POA to the lungs. For the experimental plan detailed below, established methods involving spray drying (Model B-290, Buchi) were employed to prepare POA dry powders, alone or in salt form [68], for delivery by the pulmonary route to guinea pigs using a custom built inhalation chamber [48, 69]. The POA particles shown in FIGS. 2A and 2B were fully characterized with respect to their physicochemical and aerodynamic properties. Particularly, FIG. 2A is a scanning electron microscopy image of POA (with additives), and FIG. 2B is a scanning electron microscopy image of POA leucine salt particles. All references listed herein including but not limited to all patents, patent applications and publications thereof, scientific journal articles, and database entries are incorporated herein by reference in their entireties to the extent that they supplement, explain, provide a background for, or teach methodology, techniques, and/or compositions employed herein.

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It will be understood that various details of the presently disclosed subject matter may be changed without departing from the scope of the presently disclosed subject matter. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.