Patent Application
20240366594
Described herein are compositions and methods that inhibit class I and IIb HDACs for use in inhibiting NETosis and conditions associated with NETosis.
The immune system evolved to sense microbial and endogenous cues that alter cellular or organismal homeostasis. When infection and tissue damage coincide, the immune system reacts rapidly to avoid the spread of the infection, even at the cost of an overexuberant reaction that may cause further tissue damage. Neutrophils are key players in this process (Nathan, 2002). These polymorphonuclear cells orchestrate the innate immune response by neutralizing pathogens via several effector mechanisms, which also cause tissue damage (Jaillon et al., 2013). Given their tissue damaging capabilities, neutrophils serve central roles in many inflammatory disorders and favor immune-mediated coagulation and disseminated clot formation (Gomez-Moreno et al., 2018). Central for both pathogen containment as well as development of immune-mediated disorders is the formation of neutrophil-derived extracellular traps (NETs) through a cellular process termed NETosis (Brinkmann et al., 2004; Saitoh et al., 2012; Thiam et al., 2020). Despite the key functions played by NETs against viruses, bacteria, and fungi (Thiam et al., 2020), the dysregulated formation of NETs has been linked to a variety of diseases such as acute respiratory distress syndrome (ARDS) and sepsis. ARDS and sepsis are characterized by an exaggerated elevation of pro-inflammatory cytokines in the lungs and/or bloodstream, and also by coagulopathy, thrombosis, and multiorgan dysfunction (Broggi et al., 2020; Channappanavar and Perlman, 2017; Giannis et al., 2020; Guan et al., 2020; Iba et al., 2019; Lee et al., 2020; Lucas et al., 2020; Mehta et al., 2020; Ruan et al., 2020; Shin et al., 2019; Short et al., 2014; Tang et al., 2019; Wu et al., 2019; Yang et al., 2019; Zhou et al., 2020). The central roles played by neutrophils and NETosis in driving these life-threatening immune disorders provide a strong mandate to reveal new steps in the regulation of NET induction that can be used as novel therapeutic targets.
As shown herein, class I and IIb HDACs, which belong to the family of zinc-dependent lysine deacetylases, regulate NETosis in human and mouse cells. Also, these zinc-dependent HDACs can be used as therapeutic targets in mouse models of ARDS and septic shock. In particular, the class I/IIb HDAC inhibitor ricolinostat dampens NET formation and pro-inflammatory cytokine production, reduced morbidity, and improved lung functionality in microbial pneumonia and septic shock.
Thus provided herein are methods for treating a subject who has a condition associated with NETosis, or reducing the risk of developing or delaying onset of a condition associated with NETosis, the methods comprising administering a therapeutically effective amount of an inhibitor of a class I and/or class IIb histone deacetylase (HDAC). Also provided are inhibitors of a class I and/or class IIb histone deacetylase (HDAC) for use in a method for treating a subject who has a condition associated with NETosis, or reducing the risk of developing or delaying onset of a condition associated with NETosis.
In some embodiments, the condition associated with NETosis is an autoimmune disease, a cardiovascular condition, an inflammatory condition, a condition associated with viral or bacterial infection (optionally bacterial-induced pneumonia or conditions associated with COVID-19), cytokine storm, or cancer.
In some embodiments, the autoimmune disease is Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), Type 1 diabetes mellitus (T1DM), Systemic lupus erythematosus (SLE), Rheumatoid Arthritis (RA), psoriasis, Antiphospholipid syndrome (APS), multiple sclerosis (MS), dermatomyositis (DM), polymyositis (PM), and IgG4-related autoimmune pancreatitis (AIP), or Drug-Induced Autoimmune Diseases
In some embodiments, the cardiovascular condition is small vessel vasculitis (SVV) or atherosclerosis.
In some embodiments, the inflammatory conditions is gout or Inflammatory bowel diseases (IBDs).
Also provided herein are methods for treating or reducing the risk of occurrence or reoccurrence, or delaying onset, of a cardiovascular event in a subject who has a condition associated with NETosis. The methods comprise administering a therapeutically effective amount of an inhibitor of a class I/IIb histone deacetylase (HDAC). Also provided are inhibitors of a class I/IIb histone deacetylase (HDAC), for use in a method for treating or reducing the risk of occurrence or reoccurrence, or delaying onset, of a cardiovascular event in a subject who has a condition associated with NETosis. In some embodiments, the cardiovascular event is thrombosis or myocardial infarction (MI).
In some embodiments, of the methods described herein, the subject does not have cancer, e.g., does not have a solid tumor (such as hepatocellular carcinoma) or a hematopoietic cancer, e.g., leukemia or lymphoma, e.g., multiple myeloma, acute myeloid leukemia, or B-cell lymphoma. In some embodiments, the subject does not have diabetic peripheral neuropathy.
In some embodiments, the HDAC inhibitor is an inhibitor of HDAC6.
In some embodiments, the HDAC inhibitor is entinostat, ricolinostat, citarinostat, AES-135; HDAC-IN-3 and analogues thereof; ACY-738; Abexinostat; CAY10603; WT-161; EDO-S101; UF010; Resminostat; HPOB; or CRA-026440.
In some embodiments, the subject has been identified as having a condition associated with NETosis. In some embodiments, the subject has been identified as having a level of circulating cfDNA, MPO/cfDNA conjugates and/or citH3 above a reference level. In some embodiments, the methods comprise obtaining a sample comprising blood from a subject; determining a level of circulating cfDNA, MPO/cfDNA conjugates and/or citH3 in the sample; comparing the level of circulating cfDNA, MPO/cfDNA conjugates and/or citH3 to a reference level that represents a level of circulating cfDNA, MPO/cfDNA conjugates and/or citH3 in a subject who does not have a condition associated with NETosis; and identifying a subject who has a level of circulating cfDNA, MPO/cfDNA conjugates and/or citH3 above a reference level as having a condition associated with NETosis.
In some embodiments, the subject has been identified as having a perforin mutation, as having a serum ferritin level above a threshold or reference level, or as having a level of a damage-associated molecular pattern (DAMP), optionally high mobility group box-1 (HMGB1), above a threshold or reference level.
Also provided herein are compositions comprising (i) an anti-viral drug and (ii) a small molecule inhibitor of class I and/or class IIb HDACs, and optionally further comprising a pharmaceutically acceptable carrier.
In some embodiments, the anti-viral drug is nirmatrelvir and ritonavir; sotrovimab; remdesivir; molnupiravir (Lagevrio), or an antibody, optionally bebtelovimab. In some embodiments, the HDAC inhibitor is entinostat, ricolinostat, citarinostat, AES-135; HDAC-IN-3 and analogues thereof; ACY-738; Abexinostat; CAY10603; WT-161; EDO-S101; UF010; Resminostat; HPOB; or CRA-026440. Also provided are methods of treating a viral infection in a subject, the method comprising administering a therapeutically effective amount of these compositions. In some embodiments, the viral infection is infection with a coronavirus, e.g., SARS-CoV-2.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.
Akin to transcriptional activation that is driven by local chromatin remodeling, epigenetic modifications of histones that decondense chromatin are a prerequisite for NET release (Kenny et al., 2017). In particular, histone citrullination by the peptidylarginine deiminase 4 (PAD4) has been shown to facilitate NET formation and release (Thiam et al., 2020; Wang et al., 2009). Granule-resident serine proteases, neutrophil elastase, as well as gasdermin D also participate to chromatin decondensation, nuclear expansion, and formation of NETs (Chen et al., 2018; Papayannopoulos et al., 2010; Sollberger et al., 2018). Whether other epigenetic modifications also participate in NETosis, and how they may be linked to the above-mentioned processes that alter the structure of chromatin, remains largely overlooked. Based on the importance of histone acetylation in determining chromatin structure and accessibility, we hypothesized that enzymes involved in histone acetylation also contribute to NETosis. Histone acetylation is dynamically controlled by two counteracting protein families, the histone acetyltransferases and the histone deacetylases (HDACs) as “writers” and “erasers” (Grunstein, 1997; Zhao et al., 2018). HDACs belong to two families, based on the dependency on zinc or nicotinamide adenine dinucleotide (NAD+) for their activities (Seto and Yoshida, 2014).
When infection and tissue damage coincide, the immune system potently reacts to avoid the spread of the infection, even at the cost of further damage. Neutrophils are central to this process, thanks to the release of NETs that contain and restricts viral, bacterial, as well as fungal infections (Brinkmann et al., 2004; Saitoh et al., 2012; Thiam et al., 2020). Nevertheless, excessive tissue damage caused by NET release has been often associated with immune-driven disorders (Thiam et al., 2020). Moreover, NETosis has been shown to play both protective as well as detrimental roles during tissue repair and resolution (Hahn et al., 2019; Kang et al., 2020; Schauer et al., 2014). Thus, a better understanding of the mechanisms that regulate NET induction is fundamental to develop future possible therapeutic ways of intervention to either potentiate or block the induction of NETs.
Data herein show that class I/IIb HDACs drive the release of NETs by human and mouse neutrophils. Their inhibition in mice dampens inflammation and protects against microbial pneumonia or septic shock. These data establish a new biological feature of a group of HDACs whose activity is well known to regulate gene transcription and open to the use of drugs that inhibit HDACs to restrain the tissue-damaging functions of neutrophils. Although targeting of PAD4 with the chemical compound GSK484 was previously shown to be effective in inhibiting CitH3 and NET formation, the present results demonstrate that ricolinostat, a drug currently utilized in a phase II clinical trial, can also be used to efficiently inhibit NETosis and inflammation. In multiple in vivo mouse models mimicking bacterial- or viral-induced infections and septic shock, pharmacological inhibition of HDACs ameliorated impaired lung functionality, reduced systemic inflammation, and decreased thrombosis; without wishing to be bound by theory, it appears that these effects occur via inhibition of NETosis.
Viral or bacterial-induced pneumonia and COVID-19 have a strong correlation to elevated levels of circulating neutrophils, NETs, and inflammatory cytokines that hint at a chronic inflammatory storm in patients (Mehta et al., 2020). NET formation is intimately linked to thrombosis that drives microvasculature damages in lung and other organs (Ackermann et al., 2020; Al-Samkari et al., 2020; Tang et al., 2020). Neutrophils and NETosis also participate in the most severe cases of COVID-19 (Zuo et al., 2020a, Zuo et al., 2020b). NETs infiltrate lungs of patients with severe COVID-19, and SARS-COV-2 has been shown to trigger the release of NETs that mediate lung pathology (Middleton et al., 2020; Radermecker et al., 2020; Veras et al., 2020). As dysregulated production of NETs is known to exacerbate inflammation and accelerate the pathogenesis of ARDS and sepsis caused by other coronaviruses (Blondonnet et al., 2016; Perlman and Dandekar, 2005), these findings support the hypothesis that targeting NET formation represents a viable strategy to alleviate tissue damage and hyperinflammation. Therefore, the present results imply that pharmacological inhibition of HDAC activity may be beneficial for patients with COVID-19 by blocking NETosis.
In mouse models, drugs that inhibit class I/IIb HDACs partially reduced the levels of pro-inflammatory cytokines including IL-1β, TNF, and IL-6. Overall, the present data demonstrate that zinc-dependent lysine deacetylase inhibition prevents NET formation by neutrophils, which in turn reduces systemic inflammation. Thus, provided herein are methods in which HDAC inhibition is used as a therapeutic strategy for the treatment of a variety of diseases mediated by the overproduction of NETs such as COVID-19, pulmonary diseases, autoimmune diseases (e.g., rheumatoid arthritis and systemic lupus erythematosus), diabetes, and cancers, e.g., tumor-associated inflammation, diabetes, cardiometabolic/cardiovascular diseases, anaphylaxis and pancreatitis (Arpinati et al., 2020; Lee et al., 2017; Porto and Stein, 2016; Wong et al., 2015).
The methods described herein include methods for the treatment or prophylaxis of subjects, e.g., normal subjects, or subjects who have autoimmune diseases (e.g., Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), Type 1 diabetes mellitus (T1DM), Systemic lupus erythematosus (SLE), Rheumatoid Arthritis (RA), psoriasis, Antiphospholipid syndrome (APS), multiple sclerosis (MS), dermatomyositis (DM), polymyositis (PM), and IgG4-related autoimmune pancreatitis (AIP), or Drug-Induced Autoimmune Diseases, see He et al,. Chin Med J (Engl). 2018 Jul. 5; 131(13):1513-1519; Lee et al., Autoimmun Rev. 2017 November; 16(11):1160-1173)); a cardiovascular event, e.g., thrombosis or myocardial infarction (MI), or in cardiovascular conditions such as small vessel vasculitis (SVV) or atherosclerosis; inflammatory conditions such as gout or Inflammatory bowel diseases (IBDs); complications of infection, e.g., viral or bacterial infection, e.g., viral or bacterial-induced pneumonia and consequences of COVID-19 infection, e.g., associated with cytokine storms; or cancer (see, e.g., Olsson and Cedarvall, Front Immunol. 2016; 7:373; Cedarvall and Olsson, Oncoscience. 2015; 2(11):900-901). The methods can be used, e.g., for reducing NETosis, or reducing the risk of NETosis-related conditions. Generally, the methods include administering a therapeutically effective amount of an inhibitor of class I/IIb HDACs as described herein, to a subject who is in need of, or who has been determined to be in need of (e.g., has been diagnosed with or determined to be at increased risk of developing a condition described herein), such treatment. In some embodiments, the subject does not have cancer, e.g., does not have a solid tumor (such as hepatocellular carcinoma) or a hematopoietic cancer, e.g., leukemia or lymphoma, e.g., multiple myeloma, acute myeloid leukemia, or B-cell lymphoma. In some embodiments, the subject does not have diabetic peripheral neuropathy.
Methods for identifying subjects who have a condition described herein are known in the art. Based on information known in the art a skilled healthcare practitioner could readily make a diagnosis. See, e.g., McMullin et al., Br J Haematol. 2018 Nov. 27. doi: 10.1111/bjh. 15648; Steensma et al., Blood. 2015 Jul. 2; 126(1):9-16; Heuser et al., Dtsch Arztebl Int. 2016 May 6; 113(18):317-22 Tefferi and Barbui, Am J Hematol. 2015 February; 90(2):162-73; Double and Harrison, Hematology. 2015 Mar; 20(2):119-20. In some embodiments, the subject has a perforin mutation (e.g., has been identified as having a perforin mutation) or has serum ferritin levels above a threshold.
In some embodiments, the methods can include detecting the presence of NETosis in a subject using methods known in the art (e.g., circulating cfDNA,
MPO/cfDNA conjugates and/or citH3 in serum, see Mutua and Gershwin, Clin Rev Allergy Immunol. 2021; 61(2):194-211), selecting a subject who has NETosis (e.g., based on the presence of a level of cfDNA, MPO/cfDNA conjugates and/or citH3 above a reference level, e.g., a reference level that represents a level of cfDNA, MPO/cfDNA conjugates and/or citH3 in a subject who does not have NETosis), and treating the subject using a method as described herein.
In some embodiments, the methods can include detecting the presence of a mutation in an allele of perforin in the subject, detecting serum ferritin levels and identifying a subject who has a serum ferritin level above a threshold (e.g., a threshold level that represents a level in subjects who do not have or are not at risk of developing cytokine storm syndrome, such that a level above the threshold indicates that the subject has or is at risk of developing cytokine storm syndrome), or identifying a subject who is prone to infection, e.g., subjects who are on immunosuppressants, or who are at increased risk of poor outcome, e.g., subjects who have immune disorders, are elderly (age 65 or older), or who live in in community housing facility such as a senior care home.
Subjects with perforin mutations are believed to be more susceptible to cytokine storms, which are related to NETosis; see, e.g., Schulert et al., J Infect Dis. 2016 Apr. 1; 213(7):1180-1188, found evidence of perforin pathway-related mutations in five of the 14 samples they analyzed from patients who died in the 2009 H1N1 flu outbreak. Cytokine storm syndrome was identified as a potential contributing factor in this abnormally high number of deaths in otherwise healthy individuals.
In some embodiments, the methods can include detecting the presence of damage-associated molecular patterns (DAMPs) or pro-inflammatory cytokines such as high mobility group box-1 (HMGB1), which plays a critical roles in the pathogenesis of several different inflammatory diseases (Amini et al., Rep Biochem Mol Biol. 2019 January; 7(2):204-209). HMGB1 is a sterile inflammatory molecule released from various cells during stress has been implicated in inflammation (Gonelevue et al., Stress J Vasc Res 2018; 55:244-254. The presence of DAMPs, e.g. HMGB1, above a threshold level can be used to identify subjects for treatment or prophylaxis using a method described herein.
Suitable reference values for the above can be determined using methods known in the art, e.g., using standard clinical trial methodology and statistical analysis. The reference values can have any relevant form. In some cases, the reference comprises a predetermined value for a meaningful level, e.g., a control reference level that represents a normal level of the marker, e.g., a level in an unaffected subject or a subject who is not at risk of developing a disease described herein, and/or a disease reference that represents a level associated with conditions associated with NETosis.
The predetermined level can be a single cut-off (threshold) value, such as a median or mean, or a level that defines the boundaries of an upper or lower quartile, tertile, or other segment of a clinical trial population that is determined to be statistically different from the other segments. It can be a range of cut-off (or threshold) values, such as a confidence interval. It can be established based upon comparative groups, such as where association with risk of developing disease or presence of disease in one defined group is a fold higher, or lower, (e.g., approximately 2-fold, 4-fold, 8-fold, 16-fold or more) than the risk or presence of disease in another defined group. It can be a range, for example, where a population of subjects (e.g., control subjects) is divided equally (or unequally) into groups, such as a low-risk group, a medium-risk group and a high-risk group, or into quartiles, the lowest quartile being subjects with the lowest risk and the highest quartile being subjects with the highest risk, or into n-quantiles (i.e., n regularly spaced intervals) the lowest of the n-quantiles being subjects with the lowest risk and the highest of the n-quantiles being subjects with the highest risk.
The present methods can also include administering other treatments, e.g., anti-inflammatory drugs and/or anti-viral drugs.
Inhibitors useful in the methods described herein include small molecule inhibitors of class I (HDAC1/2/3/8) and class IIb (HDAC6/10) inhibitors. Suitable inhibitors include entinostat, ricolinostat and its analog citarinostat, and AES-135 (Shouksmith et al., ACS Med Chem Lett. 2020 Jan. 9; 11(1):56-64); HDAC-IN-3 and analogues (WO/2008040934); ACY-738; Abexinostat; CAY10603; WT-161; EDO-S101; UF010; Resminostat; HPOB; or CRA-026440.
An “effective amount” is an amount sufficient to effect beneficial or desired results. For example, a therapeutic amount is one that achieves the desired therapeutic effect. This amount can be the same or different from a prophylactically effective amount, which is an amount necessary to reduce the risk of or delay onset of disease or disease symptoms. An effective amount can be administered in one or more administrations, applications or dosages. A therapeutically effective amount of a therapeutic compound (i.e., an effective dosage) depends on the therapeutic compounds selected. The compositions can be administered one from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the therapeutic compounds described herein can include a single treatment or a series of treatments.
Dosage, toxicity and therapeutic efficacy of the therapeutic compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
The methods described herein include the use of pharmaceutical compositions comprising or consisting of small molecule inhibitors of class I and/or class IIb HDACs as an active ingredient.
Pharmaceutical compositions typically include a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions, e.g., other anti-inflammatory drugs and/or anti-viral drugs. For example, provided herein are compositions comprising an anti-viral drug and a small molecule inhibitor of class I and/or class IIb HDACs. Exemplary anti-viral drugs include nirmatrelvir and ritonavir (Paxlovid); sotrovimab (Xevudy); remdesivir (Veklury); and molnupiravir (Lagevrio), and/or an antibody therapy, e.g., bebtelovimab. Such compositions are particularly useful for treating viral infections, e.g., infection with a coronavirus such as SARS-COV-2.
Pharmaceutical compositions are typically formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral, nasal (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
Methods of formulating suitable pharmaceutical compositions are known in the art, see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and the books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). For example, solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
For administration by inhalation, the compounds can be delivered in the form of an aerosol spray from a pressured container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Such methods include those described in U.S. Pat. No. 6,468,798.
Systemic administration of a therapeutic compound as described herein can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
The pharmaceutical compositions can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
In one embodiment, the therapeutic compounds are prepared with carriers that will protect the therapeutic compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations can be prepared using standard techniques, or obtained commercially, e.g., from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to selected cells with monoclonal antibodies to cellular antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.
The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
The following materials and methods were used in the examples below.
6-8 week-old female C57BL/6J (Jax 000664) and C57BL/6J-Hdac6em2Lutzy/J (Jax 029318) mice were purchased from The Jackson Laboratory. Mice were housed under specific pathogen-free conditions at Boston Children's Hospital, and all the procedures were approved under the Institutional Animal Care and Use Committee (IACUC) and operated under the supervision of the department of Animal Resources at Children's Hospital (ARCH). Sex as a biological variable was not analyzed in this work.
Human neutrophils from blood of healthy donors were purified with Polymorphoprep (Progen) according to manufacturer's instruction. Murine neutrophils were purified over a 62.5% Percoll gradient (GE Healthcare) as previously described (Broggi et al., 2017). Purified neutrophils were resuspended in DMEM medium (ThermoFisher) and plated in a 96 multi-well plate previously coated with poly-D-lysine (ThermoFisher). Plate was spin at 200×g for 1 minute without brake to facilitate neutrophils seeding.
Neutrophils were treated with indicated drugs for 1 hour and then stimulated with 1 μM PMA for 3 hours. Extracellular DNA was quantified in the supernatant with Quant-iT PicoGreen (Invitrogen).
Neutrophils pretreated with indicated HDAC inhibitors, or protein-arginine deaminase (PAD4) inhibitor GSK484, and then stimulated with 1 μM PMA for 3 hours, were fixed with equal volume of 10% formalin for 1 h at room temperature. Samples were blocked with 2% BSA in PBS for 1 hour at room temperature and stained with the anti-citrullinated histone H3 antibody overnight at 4° C., followed by anti-rabbit Alexa 568 secondary antibody. Nuclei were stained with DAPI. NETs formation was measured as induction of citrullinate histone H3 positivity and quantified with a plate-reader (Ex 568 nm, Em 603 nm). Percentage of NET induction was calculated using signal in un-treated cells as 0% of induction and signal in PMA-treated cells as 100% of induction. Alternatively, NETosis induction was quantified by imaging. Images were acquired with EVOS M7000 Imaging System (ThermoFisher Scientific). Number of citrullinated histone H3 positive cells and area of DAPI signal were quantified in multiple fields of view (FOV) using ImageJ software (NIH). For 3D nuclei reconstruction images were acquired with ZEISS 880 Fast Airyscan and analyzed with ZEISS ZEN Blue Software.
For western blotting, 2×106 neutrophils pretreated with indicated HDAC inhibitor, or protein-arginine deaminase (PAD4) inhibitor GSK484, and then stimulated with 1 μM PMA for 3 hours were lysed using RIPA buffer with protease and phosphatase inhibitors (T-2494, A.G. Scientific) and diisopropylfluorophosphate (D0879, Sigma). Immunoblotting was performed using standard molecular biology techniques. Band intensity was quantified using ImageJ software.
Neutrophils plated in a white 96 multi-well plate were pretreated with indicated HDAC inhibitor, or protein-arginine deaminase (PAD4) inhibitor GSK484 for 1 hour and then were stimulated with 1 μM PMA in presence of luminol (Cat #A8511, Sigma) and HRP (Sigma). ROS production was monitored in real-time by luminescence. Luminescence was measured every 3 minutes for 3 hours in a plate-reader analyzer (Molecular Device).
E. coli (O111) was stained with Cell Proliferation Dye eFluor™ 670 (cat #65-0840-85, Invitrogen) for 30 minutes and then opsonized with 20% mouse serum for 10 minutes. Neutrophils were stimulated with the indicated MOI for 1 hour at 37° C., washed, and then stained with anti-Ly6G PE antibody and DAPI. Ultimately, samples were acquired with BD FACS Fortessa and percentage of neutrophils positive for E. coli was quantified.
NETosis was measured in bronco-alveolar lavage fluid or lung tissue homogenates as quantity of citrullinated histone H3 with Citrullinated histone H3 ELISA kit (Cayman) and as quantity of myeloperoxidase (MPO)-DNA complexes by using a modified Cell Death Detection ELISA (Roche). Briefly, anti-myeloperoxidase antibody (ThermoFisher) was used to coat a high-binding 96 multi-well plate overnight at 4° C. Following a 1 h blocking with 2% BSA/PBS solution, samples were added for an overnight incubation at 4° C. Finally, DNA was detected with Cell Death Detection kit according to manufacture instruction. Absorbance was measured at 405 nm and MPO-DNA complex levels were quantified as normalized relative to saline.
Alternatively, neutrophils positive for citrullinated histone H3 were quantified by cytofluorimetry. Briefly, neutrophils from bronco-alveolar lavage fluid or peritoneal lavage fluid were stained for Ly6G, CD11b, CD45 and Zombie dye for 20 minutes at room temperature and then fixed with BD FACS Lysing Solution (cat #349202, BD Biosciences). Samples were washed twice with Intracellular Staining Permeabilization Wash buffer (cat #421002, Biolegend), incubated with anti-citrullinated histone H3 antibody (Abcam) 1 hour at room temperature and subsequently with PE anti-rabbit antibody for 30 minutes. Finally, samples were acquired with BD FACS Fortessa and neutrophils (CD45+/Ly6G+/CD11b+) positive for citrullinated histone H3 were quantified with FlowJo software.
6-8 weeks-old female mice were treated daily with 2.5 mg/kg of poly (I:C) for 4 days and euthanized 18 hours post the last poly(I:C) intra-tracheal instillation, as previously described (Broggi et al., 2020). Temperatures were monitored with a rectal probe. NETosis was measured in bronco-alveolar lavage fluid and lung homogenates as quantity of citrullinated histone H3 and quantity of MPO-DNA complexes. Ricolinostat (30 mg/kg/dose) and GSK484 (25 mg/kg/dose) were administered intraperitoneally at day 2, day 4 and 3 hours prior the end point. To assess lung permeability, albumin in BALF was measured with Albumin Assay Kit (ab235628, Abcam). Number of neutrophils (CD45+/Ly6G+/CD11b+) in blood, BALF and lung homogenate was quantified by cytofluorimetry using CountBright Absolute Cell Counting Beads (Thermo Scientific). Paraffin-embedded lung sections were stained with DAPI for nuclei and with anti-neutrophil antibody (Abcam) for the presence of neutrophils.
Airway hyperreactivity (AHR) was measured, as previously described (Harb et al., 2020). Anesthetized mice were exposed to doubling concentrations of aerosolized acetyl-β-methacholine (Sigma-Aldrich) by using a Buxco small-animal ventilator (Data Sciences International). The relative peak airway resistance for each methacholine dose, normalized to the saline baseline (Score=1), was calculated.
S. aureus Pulmonary Infection Model
6-8-week-old female mice were pretreated with indicated HDAC inhibitors at −1 day and −3 hours prior in vivo NETs induction by intra-tracheal instillation of 65×106 CFU of Staphylococcus aureus subsp. aureus Rosenbach (ATCC 25904). Ricolinostat was used at 30 mg/kg/dose and GSK484 at 25 mg/kg/dose. Temperatures were monitored with a rectal probe. NETosis was measured in bronco-alveolar lavage fluid as quantity of citrullinated histone H3 and quantity of MPO-DNA complexes by ELISA. Alternatively, neutrophils positive for citrullinated histone H3 were analyzed by cytofluorimetry in BALFs samples. Albumin in BALF was measured with Albumin Assay Kit (ab235628, Abcam). Number of neutrophils (CD45+/Ly6G+/CD11b+) in blood and BALF was quantified by flow cytometry using CountBright Absolute Cell Counting Beads (Thermo Scientific).
8-week-old female mice were primed with 1 mg/kg of LPS intra peritoneum (i.p.) for 5 hours and then challenged with 2 mg/kg of LPS i.p. Ricolinostat was used at 30 mg/kg/dose, GSK484 at 25 mg/kg/dose and dexamethasone at 10 mg/kg/dose. Drugs were administered i.p. at day −1 and 2 hours prior priming. Blood samples were collected 2 hours post-challenge and cytokines were measured by ELISA. The platelet concentrations and neutrophils numbers in the blood and peritoneal cavity were calculated by cytofluorimetry using CountBright Absolute Cell Counting Beads (Thermo Scientific) according to the manufacturer's instructions. NETosis was measured as quantity of neutrophils in the peritoneal lavage positive for citrullinated histone H3 by cytofluorimetry.
Results were analyzed with GraphPad Prism statistical software (version 8). One-way ANOVA and Two-way ANOVA were used to analyze statistically significant differences between the means of two or more independent groups, as indicated in the figure legends. Sample sizes for each experiment are provided in the figures and the respective legends. Asterisks were used as follows: *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.
To test the capacity of HDACs to modulate NET formation, we assembled a focused collection of class and isoform specific inhibitors of HDACs with high structural and functional diversity (Park and Kim, 2020). This included pan-HDAC inhibitors that selectively target HDAC class I (HDAC1/2/3/8), class IIa (HDAC4/5/7/9), class IIb (HDAC6/10), and class III (sirtuins), as well as an inhibitor targeting bromodomain proteins, which recognize acetylated lysine on histones. Initially, we investigated the capacity of these inhibitors to alter NETosis by measuring extracellular DNA release by primary human neutrophils activated with phorbol myristate acetate (PMA), a well-known inducer of NETs. DNA release is often used as a proxy for NETosis and, indeed, inhibition of NET induction by using the PAD4 inhibitor GSK484 resulted in significant decrease of extracellular DNA by PMA-treated cells (
A previous study suggested that pan-HDAC inhibition switches neutrophil cell death from NETosis to apoptosis (Hamam and Palaniyar, 2019). To assess whether ricolinostat reduced NETosis by favoring other types of cell death, we tested the levels of apoptosis, pyroptosis, and/or necrosis induction in PMA-treated cells exposed to either ricolinostat or GSK484. We excluded the activation of apoptosis or necroptosis under our experimental conditions (
Next, we assessed how HDAC inhibition prevents NETosis. Given that inhibition of class I/IIb HDACs decreased the release of citrullinated histone H3, we hypothesized that ricolinostat exerts its functions by increasing H3 acetylation, which in turn may prevent PAD4-dependent citrullination. Indeed, our data demonstrated that, upon ricolinostat, but not GSK484, administration, histone H3 acetylation was boosted in untreated as well as PMA-treated cells (
We next focused on the specificity of action of ricolinostat. Ricolinostat inhibits both class I and class IIb HDACs, with a preference for the latter. We, thus, compared the activity of ricolinostat with other inhibitors specific for class IIb HDACs (tubastatin A and nexturastat) as well as with tubacin, that similarly to ricolinostat targets not only class IIb HDACs but also class I HDACs (Bergman et al., 2012; Butler et al., 2010; Haggarty et al., 2003). The siderophore deferoxamine and the hydroxamate-based metalloprotease inhibitor batimastat were used as negative controls to exclude bystander activities of the drugs utilized on other divalent metals. We found that class I/IIb HDAC inhibition prevented NETosis, whereas selective class IIb targeting did not (
The conserved activity of class I/IIb HDACs in regulating NETosis in human and mouse cells prompted us to assess how HDAC inhibition with ricolinostat affects the development of pathological inflammation in vivo in murine models. To determine if the capacity of ricolinostat to inhibit NETosis as assessed in vitro also impacts viral pneumonia, we used a well-characterized mouse model that mimics the immune response to RNA respiratory viruses and that we previously successfully used to unveil some features of RNA viral infections (Broggi et al., 2020). The synthetic analog of double-stranded RNA polyinosine: polycytidylic acid (poly(I:C)) was intratracheally instilled to mice daily for 4 days to induce NET formation (
To further assess whether the capacity of ricolinostat to prevent NETosis can be used against ARDS, we employed a bacterial lung pneumonia mouse model based on the intratracheal injection of S. aureus (
Finally, we investigated whether inhibition of class I/IIb HDACs dampens systemic inflammation in a mouse model of septic shock. Septic shock is characterized by systemic inflammation and disseminated intravascular coagulation (van der Poll et al., 2017), and neutrophils play a critical role in driving this lethal syndrome. We hypothesized that inhibition of NET formation using ricolinostat may yield beneficial effects in reducing sepsis-associated symptoms. Thus, we employed a well-established lipopolysaccharide (LPS)-driven model of septic shock (Zanoni et al., 2017) in mice treated, or not, with ricolinostat or GSK484, as a comparison (
Since dexamethasone is widely used to dampen inflammation, we also compared the therapeutic effects of ricolinostat with dexamethasone. We did not observe a significant difference in platelet numbers when comparing the dexamethasone-treated group with the group treated with LPS only (
Bergman, J.A., Woan, K., Perez-Villarroel, P., Villagra, A., Sotomayor, E.M., and Kozikowski, A.P. (2012). Selective Histone Deacetylase 6 Inhibitors Bearing Substituted Urea Linkers Inhibit Melanoma Cell Growth. J Med Chem 55, 9891-9899.
Broggi, A., Tan, Y., Granucci, F., and Zanoni, I. (2017). IFN-lambda suppresses intestinal inflammation by non-translational regulation of neutrophil function. Nat Immunol 18, 1084-1093.
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application claims the benefit of U.S. Provisional Application Ser. No. 63/194, 153, filed on May 27, 2021. The entire contents of the foregoing are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/072609 | 5/27/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63194153 | May 2021 | US |
