Patent Application
20160220710
Provided herein are biomimetic microcrystalline forms of drugs. In particular, provided herein are microcrystalline drug formulations for delivery to macrophages and treatment of disease.
The lung is vulnerable to many inflammatory disorders as it is the only internal organ that is exposed constantly to the external environment. Consequently, respiratory diseases cause an immense worldwide health burden. According to the World Health Organization (WHO) and Forum of International Respiratory Societies (FIRS), it is estimated that ˜15% of the world's population suffers from chronic respiratory conditions, including 235 million people suffering from asthma, more than 200 million from chronic obstructive pulmonary disease (COPD) and these numbers are growing every year. In the case of acute respiratory distress syndrome (ARDS), 200,000 Americans per year are affected, with a mortality rate of 40% but no available therapeutic drug available. Healthcare costs for respiratory diseases are an increasing burden on the economies of all countries, but respiratory diseases are rarely on the public health agenda.
Respiratory diseases, such as ARDS, COPD and asthma, are caused by an uncontrolled inflammatory response characterized by dysregulated pro- and anti-inflammatory mediators and increased numbers and/or altered activation of immune cells, including macrophages. Key inflammatory mediators are the pro-inflammatory cytokines tumor necrosis factor α (TNFα) and interleukin-1 (IL-1α and IL-1β), which are required for the initiation and activation of the immune response, and the anti-inflammatory cytokine interleukin-1 receptor antagonist (IL-1RA), which counteracts IL-1 by competitively binding to IL-1 receptor to block signal transduction and resolve inflammation. Continued dysregulation of TNFα and IL-1 expression is a hallmark of chronic inflammatory disorders of the lung. Macrophages play a critical role in the initiation and resolution of lung inflammation, and importantly, one of the major producers of TNFα and IL-1RA.
Existing anti-inflammatory drugs that act by blocking TNFα activity (e.g., etanercept) or by blocking IL-1 via IL-1RA (e.g., anakinra) are currently undergoing preclinical studies or clinical trials as single agents for the potential treatments for COPD, idiopathic pulmonary fibrosis, asthma, and acute respiratory distress syndrome amongst others. However, both TNFα inhibitors and IL-1RA are soluble agents that when systemically injected are poised to affect the whole body indiscriminately and can lead to serious side effects, including increased susceptibility to infection and sepsis.
Accordingly, macrophage-targeted therapeutic strategies that aim to modulate the intracellular signaling pathways affecting TNFα and IL-1RA balance are needed.
Provided herein are microcrystalline forms of drugs. In particular, provided herein are microcrystalline drug formulations for delivery to macrophages and treatment of disease.
For example, in some embodiments, the present disclosure provides compositions comprising a biomimetic crystal of a pharmaceutical agent. In some embodiments, the biomimetic crystal is a pure drug crystal or in complex with a counterion or cell-stabilizing agent as a salt, hydrate, solvate or a cocrystal. In some embodiments, the biomimetic crystal has an orthorhombic, triclinic, or monoclinic crystal structure. In some embodiments, the crystal has a needle, cube, blade, prism, or rhomboid habit. In some embodiments, the small molecule pharmaceutical agent is clofazimine. In some embodiments, the orthorhombic crystalline structural form has the configuration space group Pbca. In some embodiments, the salt has the crystal structure shown in the Figures. In some embodiments, the density of the crystals is between 1.25-1.4 g/ml and the crystals have a size of 0.001-20 μm in each dimension. In some embodiments, the salt comprises chloride. In some embodiments, the cell stabilizing agent is hydrochloride. In some embodiments, the salt further comprises one or more additional ions or cell stabilizing agents (e.g. one or more (e.g., two or more) of hydrochloride, hydrobromide, hydroiodide, hydrogen sulfate, sulfate, hydrogen phosphate, phosphate, carbonate, hydrogen carbonate, formate, gluconate, lactate, pyruvate, nitrite, glutarate, tartarate, benzoate, sulfate, fumarate, benzenesulfonate, tosylate, galacturonate, acetate, citrate, nitrate, oxalate, succinate, and maleate). In some embodiments, the ratio of pharmaceutical agent to ion is approximately 0.1 to 2.0. In some embodiments, the composition is provided as a crystal hydrate, crystal salt, or cocrystal. In some embodiments, the composition further comprises a lipid. In some embodiments, the pharmaceutical agent is encapsulated by a liposome comprising the lipid. In some embodiments, the lipid is one or more of phosphatidylcholine, cholesterol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, sphingomyelin, cardiolipin, dioleoylphosphatidylglycerol (DOPG), diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, diacylglycerols, dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), and dioleoylphosphatidylserine (DOPS), diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoyl phosphatidylethanolamines, N-succinyl phosphatidylethanolamines, N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), lecithin, lysolecithin, phosphatidylethanolamine, lysophosphatidylethanolamine, dioleoylphosphatidylethanolamine (DOPE), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), palmitoyloleoyl-phosphatidylethanolamine (POPE) palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), di stearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleyolphosphatidylglycerol (POPG), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, palmitoyloleoyl-phosphatidylethanolamine (POPE), 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerolricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethyl ammonium bromide, polyethylene glycol (PEG), or PEG modified lipids. In some embodiments, the lipid or other encapsulating agent is modified to comprise a targeting agent (e.g., including but not limited to, antibodies, mannose, folate, or transferrin). In some embodiments, the composition further comprises one or more of a non-ionic surfactant, a niosome, a polymer, a protein, or a carbohydrate.
Further embodiments provide a method of treating or preventing a disease in a subject, comprising: administering any of the aforementioned compositions to a subject diagnosed with or suspected of having a disease. In some embodiments, the administering reduces or eliminates signs and/or or symptoms of the disease. In some embodiments, the disease is asthma, bronchiolitis, bronchiolitis obliterans, chronic obstructive pulmonary disease (COPD), bronchitis, emphysema, hypersensitivity pneumonitis, idiopathic pulmonary fibrosis, pneumoconiosis, silicosis, meningitis, sepsis, malaria, rheumatoid osteoarthritis, psoriasis, acute respiratory disease syndrome, inflammatory bowel disease, multiple sclerosis, joint inflammation, reactive arthritis, hay fever, atherosclerosis, rheumatoid arthritis, bursitis, gouty arthritis, osteoarthritis, polymyalgia rheumatic arthritis, septic arthritis, infectious arthritis, asthma, autoimmune diseases, chronic inflammation, chronic prostatitis, glomerulonephritis, nephritis, inflammatory bowel diseases, pelvic inflammatory disease, reperfusion injury, transplant rejection, vasculitis, myocarditis, colitis, appendicitis, peptic ulcer, gastric ulcer, duodenal ulcer, peritonitis, pancreatitis, ulcerative colitis, seudomembranous colitis, acute colitis, ischemic colitis, diverticulitis, epiglottitis, achalasia, cholangitis, cholecystitits, hepatitis, Crohn's disease, enteritis, Whipple's disease, allergy, anaphylactic shock, immune complex disease, organ ischemia, reperfusion injury, organ necrosis, hay fever, sepsis, septicemia, endotoxic shock, cachexia, hyperpyrexia, eosinophilic granuloma, granulomatosis, sarcoidosis, septic abortion, epididymitis, vaginitis, prostatitis, urethritis, bronchitis, emphysema, rhinitis, pneumonitits, pneumoultramicroscopic silicovolcanoconiosis, alvealitis, bronchiolitis, pharyngitis, pleurisy, sinusitis, influenza, respiratory syncytial virus infection, HIV infection, hepatitis B virus infection, hepatitis C virus infection, herpes virus infection disseminated bacteremia, Dengue fever, candidiasis, malaria, filariasis, amebiasis, hydatidcysts, burns, dermatitis, dermatomyositis, sunburn, urticaria, Warts, Wheals, vasulitis, angiitis, endocarditis, arteritis, atherosclerosis, thrombophlebitis, pericarditis, myocarditis, myocardial ischemia, periarteritis nodosa, rheumatic fever, Alzheimer's disease, coeliac disease, congestive heart failure, adult respiratory distress syndrome, meningitis, encephalitis, multiple sclerosis, cerebral infarction, cerebral embolism, Guillame-Barre syndrome, neuritis, neuralgia, spinal cord injury, paralysis, uveitis, arthritides, arthralgias, osteomyelitis, fasciitis, Paget's disease, gout, periodontal disease, rheumatoid arthritis, synovitis, myasthenia gravis, thyroiditis, systemic lupus erythematosis, Goodpasture's syndrome, Behcet's syndrome, allograft rejection, graft-versus-host disease, Type I diabetes, Type II diabetes, ankylosing spondylitis, Berger's disease, Reiter's syndrome, Hodgkin's disease, ileus, hypertension, irritable bowel syndrome, myocardial infarction, sleeplessness, anxiety, local inflammation, or stent thrombosis. In some embodiments, the disease is caused by infection by a microorganism, e.g., Staphylococcus aureus, Streptococcus, Streptococcus pneumonia, Neisseria gonorrhoeae, Mycobacterium tuberculosis, Borrelia burgdorferi, or Haemophilus influenza.
In some embodiments, the composition is targeted to macrophages of the subject (e.g., the composition is phagocytized by the macrophage). In some embodiments, the composition has a biological effect in the subject (e.g., one or more of inhibition of TNFα production, enhancement of IL-1RA production, or downregulation of toll-like receptor expression). In some embodiments, the administering is parentally, via inhalation, or nasally. For example, in some embodiments, the administering is to the lung via inhaler or nebulizer.
Additional embodiments provide the use of any of the aforementioned compositions to treat a disease in a subject.
Other embodiments provide a method of identifying inflammation in a joint of a subject, comprising: a) administering any of the aforementioned compositions or clofazimine to the subject; and b) performing photo-Acoustic Tomography (PAT) of a joint of the subject to identify the presence of the composition in the joint, wherein the presence of the composition in said joint is indicative of inflammation in the joint. In some embodiments, the method further comprises the step of imaging said joint using ultrasound. In some embodiments, the joint is, for example, a knee joint, a finger joint, a toe joint, a hip joint, or an elbow joint. In some embodiments, the presence of inflammation in said joint is indicative of arthritis in said joint. In some embodiments, the composition or clofazimine treats the inflammation in the joint.
Still other embodiments provide the use of any of the aforementioned compositions or clofazimine as a PAT contrast agent for imaging a joint.
Additional embodiments are described herein.
As used herein, the term “subject” refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms “subject” and “patient” are used interchangeably herein in reference to a human or non-human mammal subject.
As used herein, the term “diagnosed,” as used herein, refers to the recognition of a disease by its signs and symptoms (e.g., resistance to conventional therapies), or genetic analysis, pathological analysis, histological analysis, and the like.
As used herein, the term “effective amount” refers to the amount of a compound (e.g., a compound of the present disclosure) sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages and is not limited to a particular formulation or administration route.
As used herein, the term “co-administration” refers to the administration of at least two agent(s) (e.g., a compound of the present disclosure) or therapies to a subject. In some embodiments, the co-administration of two or more agents/therapies is concurrent. In some embodiments, a first agent/therapy is administered prior to a second agent/therapy. Those of skill in the art understand that the formulations and/or routes of administration of the various agents/therapies used may vary. The appropriate dosage for co-administration can be readily determined by one skilled in the art. In some embodiments, when agents/therapies are co-administered, the respective agents/therapies are administered at lower dosages than appropriate for their administration alone. Thus, co-administration is especially desirable in embodiments where the co-administration of the agents/therapies lowers the requisite dosage of a known potentially harmful (e.g., toxic) agent(s).
As used herein, the term “pharmaceutical composition” refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo, in vivo or ex vivo.
As used herein, the term “pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions (e.g., such as an oil/water or water/oil emulsions), and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants. (See e.g., Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, Pa., (1975)).
As used herein, the term “cell stabilizing agent” refers to an agent (e.g., ion, lipid, or other agent) in complex with a biocrystalline mimetic of pharmaceutical agent. In some embodiments, the cell stabilizing agent stabilizes the complex in vivo.
As used herein, the term “sample” is used in its broadest sense. In one sense, it is meant to include a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include blood products, such as plasma, serum and the like. Environmental samples include environmental material such as surface matter, soil, water and industrial samples. Such examples are not however to be construed as limiting the sample types applicable to the present disclosure.
As used herein, the terms “purified” or “to purify” refer, to the removal of undesired components from a sample. As used herein, the term “substantially purified” refers to molecules that are at least 60% free, at least 65% free, at least 70% free, at least 75% free, at least 80% free, at least 85% free, at least 90% free, at least 95% free, at least 96% free, at least 97% free, at least 98% free, at least 99% free, or 100% free from other components with which they usually associated.
As used herein, the term “modulate” refers to the activity of a compound (e.g., a compound of the present disclosure) to affect (e.g., to promote or retard) an aspect of cellular function.
As used herein, the phrase “in need thereof” means that the subject has been identified as having a need for the particular method or treatment. In some embodiments, the identification can be by any means of diagnosis. In any of the methods and treatments described herein, the subject can be in need thereof. In some embodiments, the subject is in an environment or will be traveling to an environment in which a particular disease, disorder, condition, or injury is prevalent.
Provided herein are microcrystalline forms of drugs. In particular, provided herein are microcrystalline drug formulations for delivery to macrophages and treatment of disease.
The elucidation of molecular mechanisms influencing the solubility of poorly soluble chemical agents in different cells, tissues and organs of mammals is interesting from a fundamental chemical and biological perspective. Cells are able to eliminate soluble chemical agents via metabolism and facilitated or active transport across the plasma membrane. Moreover, in the case of foreign, insoluble particles, phagocytic cells of the immune system are especially equipped to actively ingest these particles and isolate them from the rest of the organism. However, for poorly soluble compounds, that can exist both as soluble and insoluble forms within the cells of an organism, the mechanisms controlling the bioaccumulation and distribution of soluble and insoluble forms of these agents in the different cells and organs of the body are not known.
Many FDA-approved drugs (e.g., clofazimine, amiodarone, azithromycin, chloroquine, gefitinib) fall within the class of poorly soluble compounds that are actively sequestered within macrophages (Ohkuma, et al., J. Cell Biol. 1981, 90, 656-664; Poole, et al., J. Cell Biol. 1981, 90, 665-669; Maxfield, et al., J. Cell Biol. 1982, 95, 676-681; Quaglino, et al., Am. J. Physiol. Lung Cell. Mol. Physiol. 2004, 287, 438-447; Bergman, et al., Int. J. Pharm. 2007, 341, 134-142; Gladue, et al., Antimicrob. Agents Chemother. 1989, 33, 277-282.). Elucidating the mechanisms affecting their solubility and accumulation inside cells is relevant to understanding drug toxicity, disposition and efficacy (Fu, et al., Nat. Chem. 2014, 6, 614-622). Particularly, weakly basic molecules have been implicated to be accumulated via the lysosomal pathway in macrophages due to the decrease in pH of the vesicles that contain the drug (Ohkuma et al., supra). Research into mechanisms responsible for in vivo drug bioaccumulation and retention has been very sparse.
CFZ (Harbeck, et al., Ann. Pharmacother. 1999, 33, 250; Levy, L. Am. J. Trop. Med. Hyg. 1974, 23, 1097-1109; Aplin, et al., Experientia 1975, 31, 468-469; McDougall, et al., Br. J. Dermatol. 1980, 102, 227-230; McDougall, Int J Lepr. Other Mycobact. Dis. 1974, 42, 1-12) is an FDA-approved riminophenazine antibiotic that has been remarkably effective against mycobacterial infections such as leprosy (Tolentino, et al., Int J Lepr. Other Mycobact. Dis. 1974, 42, 416-418; Karuru, et al. Lepr. Rev. 1970, 41, 83-88; Leiker, et al., Lepr. Rev. 1971, 42, 125-130) and mycobacterium avium infections in AIDS patients (Rensburg, et al., Antimicrob. Agents Chemother. 1992, 36, 2729-2735; Reddy, et al., J. Antimicrob. Chemother. 1999, 43, 615-623). It has also attracted attention because of its anti-inflammatory (Cholo, et al., J. Antimicrob. Chemother. 2012, 67, 290-298; Helmy, et al., Lepr. Rev. 1972, 42, 162-177) and immunomodulatory (Ren, et al., PLoS One 2008, 3, e4009) properties, and it is especially used for treating leprotic skin inflammations (erythema leprosum nodosum) (Cholo et al., supra; Helmy et al., supra; Barry, et al., Lepr. Rev. 1965, 36, 3-7; Barry, et al., Nature 1957, 179, 1013-1015).
The chemical structure of CFZ (3-(p-chloroanilino)-10-(p-chlorophenyl)-2, 10-dihydro-2-isopropyliminophenazine) is
Derivatives of CFZ are described, for example, in Franzblau et al., Antimicrob Agents Chemother 1988; 32:1583-5; Jagannath C, et al., Am J Respir Crit Care Med 1995; 151:1083-6; O'Sullivan J F, et al., Health Cooperation Papers 1992; 12:191-7; and O'Connor R, et al., J Chromatogr B Biomed Appl 1996; 681:307-15; each of which is herein incorporated by reference in its entirety.
Accordingly, provided herein are compositions and method for the synthesis, characterization and composition of biomimetic, anti-inflammatory macrophage-targeted, microcrystalline drug formulations (e.g., of CFZ). Alveolar macrophages are primary sites for the anti-inflammatory response in the lung. During or after the inflammation, biomimetic drug crystal formulations are inhaled by the patient or can be administered via intravenous/systemic injections/oral route leading to active uptake by macrophages through phagocytosis. This targeted accumulation causes the macrophages to become more efficient at their anti-inflammatory activity via both down-regulation of TNFα as well as up-regulation of IL-1RA. Both acute as well as chronic disorders are targeted through this approach. Since these are stable drug formulations, their effect is sustained for a long-period of time without the need for repeated dosages. Moreover, since they are targeted to macrophages, their effect is localized to the site of inflammation (e.g., lung or joints), thereby reducing any systemic drug side effects.
Provided herein are biomimetic drug microcrystals of small molecule pharmaceutical agents (e.g., CFZ). In some embodiments, the agents are present as salts comprising counterions. In some embodiments, compositions mimic the natural solid state in terms of the drug protonation state, counterions, salt form or crystal polymorph of the drugs (e.g., a biomimetic of a naturally occurring crystal form present in the CLDIs that accumulate in macrophages of drug-treated animals upon long term oral dosing).
The present disclosure is illustrated with CFZ formulations. However, the present disclosure is not limited to CFZ. Crystalline forms of other agents (e.g., agents that accumulate as CLDIs in vivo) are specifically contemplated by the present disclosure.
In some embodiments, active agents are present as a salt with a counterion. For example, in some embodiments, CFZ is present as a salt is positively charged and formulations comprise one or more anions. In some embodiments, the anion is chloride. In some embodiments, the anion is one or more of bromide, iodide, sulfate, phosphate, carbonate, acetate, citrate, nitrate, oxalate, succinate, or maleate. In some embodiments, the ratio of drug:counterion is between approximately 0.1-2.0, although other ratios find use in some embodiments.
Crystalline forms of active agents are formed using any suitable method. For example, in some embodiments, the active agent is dissolved in a solvent (e.g., methanol) and equal volumes of anti-solvents (e.g., comprising counterion) are added to obtain drug crystals. The supernatant is then removed, the crystals are washed, and optionally lyophilized. In some embodiments, crystals are generated by mechanical milling and homogenization.
In some embodiments, crystalline formulations exhibit specific dichroism properties. The interaction of CLDIs with polarized light is highly distinct from other pure CFZ crystals. Such interactions can be quantitatively measured using linear dichroism, an optical parameter that measures optical anisotropy within solid structures of transmittance of light. In some embodiments, dichroic ratios of the formulations described herein at 546 nm and 623 nm (LD623/LD546) is approximately 0.5±0.1. In some embodiments, crystals have an orthorhombic crystal structure (space group: Pbca) with a denser molecular packing compared to triclinic crystals. In some embodiments, crystalline formulations of CFZ described herein have a bulk density in the range of 1.33-1.4 g/ml. In some embodiments, crystalline formulations of CFZ described have a size range between 0.001-20 μm in one and/or two dimensions.
In some embodiments, compositions further comprise one or more lipids. In some embodiments, the lipids are present as a liposome that encapsulates the pharmaceutical agent (e.g., to mimic cellular membranes). For example, in some embodiments, biomimetic forms of the crystal are formed using a remote loading of drugs via ammonium salt method or direct lipid encapsulation of ammonium salt precipitated crystal salt of drug (See e.g., Ceh et al., Langmuir, 1995, 11 (9), pp 3356-3368).
In some embodiments, compositions are provides as crystal hydrates, crystal solvates, or cocrystals.
In some embodiments, the pharmaceutical agent is encapsulated in a niosome (See e.g., Moghassemi et al, Journal of Controlled Release, Volume 185, 10 Jul. 2014, Pages 22-36). Niosomes are a class of molecular cluster formed by self-association of non-ionic surfactants in an aqueous phase.
In some embodiments, the lipid (e.g., phospholipid) structures surrounding crystalline pharmaceutical agent is tailored to the target organ/tissue lipid composition. For example, the natural lipid composition in the lungs is rich in choline lipids. Hence, in some embodiments, synthetic lipids comprising phosphatidylcholines are used in formulating compositions for delivery to the lung. The specific composition and morphology of the formulation is modulated through temperature and concentrations of lipid as well as drug:lipid ratio (See e.g., Keswani et al., Mol Pharm. 2013 May 6; 10(5):1725-35; herein incorporated by reference in its entirety).
In some embodiments, lipids are functionalized to aid in phagocytosis by macrophages. For example, in some embodiments, compositions are enveloped with mannose-conjugated phospholipids that are internalized via the CD206/CD205 receptor on macrophages.
In some embodiments, at least one (or some) of the lipids is/are amphipathic lipids, defined as having a hydrophilic and a hydrophobic portion (typically a hydrophilic head and a hydrophobic tail). The hydrophobic portion typically orients into a hydrophobic phase (e.g., within the bilayer), while the hydrophilic portion typically orients toward the aqueous phase (e.g., outside the bilayer, and possibly between adjacent apposed bilayer surfaces). The hydrophilic portion may comprise polar or charged groups such as carbohydrates, phosphate, carboxylic, sulfato, amino, sulfhydryl, nitro, hydroxy and other like groups. The hydrophobic portion may comprise apolar groups that include without limitation long chain saturated and unsaturated aliphatic hydrocarbon groups and groups substituted by one or more aromatic, cyclo-aliphatic or heterocyclic group(s). Examples of amphipathic compounds include, but are not limited to, phospholipids, aminolipids and sphingolipids.
In some embodiments, the lipids are phospholipids. Phospholipids include without limitation phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, and the like. It is to be understood that other lipid membrane components, such as cholesterol, sphingomyelin, cardiolipin, etc. may be used.
The lipids may be anionic and neutral (including zwitterionic and polar) lipids including anionic and neutral phospholipids. Neutral lipids exist in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, dioleoylphosphatidylglycerol (DOPG), diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides and diacylglycerols. Examples of zwitterionic lipids include without limitation dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), and dioleoylphosphatidylserine (DOPS). An anionic lipid is a lipid that is negatively charged at physiological pH. These lipids include without limitation phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoyl phosphatidylethanolamines, N-succinyl phosphatidylethanolamines, N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids.
Collectively, anionic and neutral lipids are referred to herein as non-cationic lipids. Such lipids may contain phosphorus but they are not so limited. Examples of non-cationic lipids include lecithin, lysolecithin, phosphatidylethanolamine, lysophosphatidylethanolamine, dioleoylphosphatidylethanolamine (DOPE), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), palmitoyloleoyl-phosphatidylethanolamine (POPE) palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), di stearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleyolphosphatidylglycerol (POPG), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, palmitoyloleoyl-phosphatidylethanolamine (POPE), 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, and cholesterol.
Additional nonphosphorous containing lipids include stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerolricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethyl ammonium bromide and the like, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, and cerebrosides. Lipids such as lysophosphatidylcholine and lysophosphatidylethanolamine may be used in some instances. Noncationic lipids also include polyethylene glycol-based polymers such as PEG 2000, PEG 5000 and polyethylene glycol conjugated to phospholipids or to ceramides (referred to as PEG-Cer).
In some embodiments, lipids are cationic lipids (e.g., those described herein).
In some instances, modified forms of lipids may be used including forms modified with detectable labels such as fluorophores. In some instances, the lipid is a lipid analog that emits signal (e.g., a fluorescent signal). Examples include without limitation 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR) and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine (DiD).
In some embodiments, the lipids are biodegradable in order to allow release of encapsulated agent in vivo and/or in vitro. Biodegradable lipids include but are not limited to 1,2-dioleoyl-sn-glycero-3-phosphocholine (dioleoyl-phosphocholine, DOPC), anionic 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phospho-(1′-rac-glycerol) (dioleoyl-phosphoglycerol, DOPG), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (distearoyl-phosphoethanolamine, DSPE). Non-lipid membrane components such as cholesterol may also be incorporated.
One or more of the lipids may be functionalized lipids. In some embodiments, the reactive group is one that will react with a crosslinker (or other moiety) to form crosslinks between such functionalized lipids. The reactive group may be located anywhere on the lipid that allows it to contact a crosslinker and be crosslinked to another lipid in an adjacent apposed bilayer. In some embodiments, it is in the head group of the lipid, including for example a phospholipid. An example of a reactive group is a maleimide group. Maleimide groups may be crosslinked to each other in the presence of dithiol crosslinkers such as but not limited to dithiolthrietol (DTT). An example of a functionalized lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl) butyramide, referred to herein as MPB. Another example of a functionalized lipid is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)2000] (also referred to as maleimide-PEG 2k-PE). Another example of a functionalized lipid is dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal).
It is to be understood that the disclosure contemplates the use of other functionalized lipids, other functionalized lipid bilayer components, other reactive groups, and other crosslinkers. In addition to the maleimide groups, other examples of reactive groups include but are not limited to other thiol reactive groups, amino groups such as primary and secondary amines, carboxyl groups, hydroxyl groups, aldehyde groups, alkyne groups, azide groups, carbonyls, haloacetyl (e.g., iodoacetyl) groups, imidoester groups, N-hydroxysuccinimide esters, sulfhydryl groups, pyridyl disulfide groups, and the like.
Functionalized and non-functionalized lipids are available from a number of commercial sources including Avanti Polar Lipids (Alabaster, Ala.).
In some embodiments, compositions further comprise one or more additional agents. Examples include, but are not limited to, polymers, proteins, carbohydrates, or other natural or artificial molecular components that serve to enhance the targeting or activity of the active agent (e.g., by promoting the binding to or phagocytosis by alveolar macrophages, or by slowing down the degradation/decomposition/clearance by macrophages in other sites of the body).
Embodiments of the present disclosure provide methods of using the aforementioned crystalline drug formulations (e.g., CFZ) in the treatment of disease (e.g., respiratory or inflammatory disease). The present disclosure is not limited to particular inflammatory diseases. Exemplary diseases are described herein.
The compositions described herein find use in the treatment of a variety of acute and chronic respiratory disease. Examples include, but are not limited to, asthma, bronchiolitis, bronchiolitis obliterans, chronic obstructive pulmonary disease (COPD), bronchitis, emphysema, hypersensitivity pneumonitis, idiopathic pulmonary fibrosis, pneumoconiosis, or silicosis.
Further example of inflammatory disease include, but are not limited to acute bacterial or viral infection e.g. meningitis, sepsis, malaria or chronic inflammatory diseases such as rheumatoid osteoarthritis, psoriasis, acute respiratory disease syndrome, inflammatory bowel disease (ulcerative colitis and Crohn's disease), multiple sclerosis, etc. In some embodiments, the inflammatory disease is local inflammation (e.g., at local sites such as eyes/cornea/conjunctiva, sclera, vitreous humor etc.).
In some embodiments, the compositions described herein find use in the treatment of joint inflammation (either acute or chronic) induced due to infection of any other organs via the causative microorganisms. These conditions can also be categorized as septic arthritis or infectious arthritis or inflammatory arthritis. In some embodiments, infectious arthritis is caused by Staphylococcus aureus, Streptococcus, Streptococcus pneumonia, Neisseria gonorrhoeae, Mycobacterium tuberculosis, Borrelia burgdorferi, or Haemophilus influenza.
The compositions further find use in the treatment of arthritis. Arthritis also develops in people who have infections that do not involve the bones or joints, such as infections of the genital organs or digestive organs or ocular regions. This type of arthritis is a reaction to that infection and so is called reactive arthritis. In reactive arthritis, the joint is inflamed but not actually infected.
Additional types of inflammatory disorders that may be treated as described herein include a variety of disease states, including diseases such as hay fever, atherosclerosis, arthritis (rheumatoid, bursitis, gouty arthritis, osteoarthritis, polymyalgia rheumatic, etc.), asthma, autoimmune diseases, chronic inflammation, chronic prostatitis, glomerulonephritis, nephritis, inflammatory bowel diseases, pelvic inflammatory disease, reperfusion injury, transplant rejection, vasculitis, myocarditis, colitis, appendicitis, peptic ulcer, gastric ulcer, duodenal ulcer, peritonitis, pancreatitis, ulcerative colitis, seudomembranous colitis, acute colitis, ischemic colitis, diverticulitis, epiglottitis, achalasia, cholangitis, cholecystitits, hepatitis, Crohn's disease, enteritis, Whipple's disease, allergy, anaphylactic shock, immune complex disease, organ ischemia, reperfusion injury, organ necrosis, hay fever, sepsis, septicemia, endotoxic shock, cachexia, hyperpyrexia, eosinophilic granuloma, granulomatosis, sarcoidosis, septic abortion, epididymitis, vaginitis, prostatitis, urethritis, bronchitis, emphysema, rhinitis, pneumonitits, pneumoultramicroscopic silicovolcanoconiosis, alvealitis, bronchiolitis, pharyngitis, pleurisy, sinusitis, influenza, respiratory syncytial virus infection, HIV infection, hepatitis B virus infection, hepatitis C virus infection, herpes virus infection disseminated bacteremia, Dengue fever, candidiasis, malaria, filariasis, amebiasis, hydatidcysts, burns, dermatitis, dermatomyositis, sunburn, urticaria, Warts, Wheals, vasulitis, angiitis, endocarditis, arteritis, atherosclerosis, thrombophlebitis, pericarditis, myocarditis, myocardial ischemia, periarteritis nodosa, rheumatic fever, Alzheimer's disease, coeliac disease, congestive heart failure, adult respiratory distress syndrome, meningitis, encephalitis, multiple sclerosis, cerebral infarction, cerebral embolism, Guillame-Barre syndrome, neuritis, neuralgia, spinal cord injury, paralysis, uveitis, arthritides, arthralgias, osteomyelitis, fasciitis, Paget's disease, gout, periodontal disease, rheumatoid arthritis, synovitis, myasthenia gravis, thyroiditis, systemic lupus erythematosis, Goodpasture's syndrome, Behcet's syndrome, allograft rejection, graft-versus-host disease, Type I diabetes, Type II diabetes, ankylosing spondylitis, Berger's disease, Reiter's syndrome, Hodgkin's disease, ileus, hypertension, irritable boWel syndrome, myocardial infarction, sleeplessness, anxiety and stent thrombosis.
The compositions described herein provide a variety of advantages over the delivery of existing formulations of CFZ. Examples include, but are not limited to, enhanced anti-inflammatory response compared to soluble formulations, dual cell-targeted anti-inflammatory response by increasing endogeneous IL-1RA production and reduction in TNF-α levels, enhanced and specific delivery to alveolar macrophages, reduced/No toxicity, and sustained controlled release delivery of therapeutics, thus not requiring repeated medication resulting in better quality of care and patient adherence.
In some embodiments, administration of microcrystalline formulations of drugs (e.g., CFZ) that target macrophages results in several molecular effects. In some embodiments, one or more of the following physiological effects is observed following administration. Inhibition of TNFα production: The active phagocytosis and intracellular compartmentalization of CLDIs by macrophages results in the dampening of NF-κB activation and TNFα release in response to TLR2 and TLR4 agonists. In addition, enhancement of IL-1RA production is also a result of the formulations described herein. The phagocytosis of CLDIs augments the activation of PI-3K/Akt signaling pathways, which leads to enhanced IL-1RA production and release. Downregulation of Toll-like receptors (TLR): The active phagocytosis of CLDIs by macrophages results in the specific downregulation of cell surface TLR2 and TLR4, which play important roles in modulating immune responses.
Pharmaceutical formulations include those suitable for oral, rectal, nasal, topical (including buccal and sub-lingual), vaginal or parenteral (including intramuscular, sub-cutaneous and intravenous) administration or in a form suitable for administration by inhalation, insufflation or by a transdermal patch. Transdermal patches dispense a drug at a controlled rate by presenting the drug for absorption in an efficient manner with minimal degradation of the drug. Typically, transdermal patches comprise an impermeable backing layer, a single pressure sensitive adhesive and a removable protective layer with a release liner. One of ordinary skill in the art will understand and appreciate the techniques appropriate for manufacturing a desired efficacious transdermal patch based upon the needs of the artisan.
The compounds described herein, optionally together with a conventional adjuvant, carrier, or diluent, may thus be placed into the form of pharmaceutical formulations and unit dosages thereof and in such form may be employed as solids, such as tablets or filled capsules, or liquids such as solutions, suspensions, emulsions, elixirs, gels or capsules filled with the same, all for oral use, in the form of suppositories for rectal administration; or in the form of sterile injectable solutions for parenteral (including subcutaneous) use. Such pharmaceutical compositions and unit dosage forms thereof may comprise conventional ingredients in conventional proportions, with or without additional active compounds or principles and such unit dosage forms may contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage range to be employed.
For oral administration, the pharmaceutical composition may be in the form of, for example, a tablet, capsule, suspension or liquid. The pharmaceutical composition is preferably made in the form of a dosage unit containing a particular amount of the active ingredient. Examples of such dosage units are capsules, tablets, powders, granules or a suspension, with conventional additives such as lactose, mannitol, corn starch or potato starch; with binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators such as corn starch, potato starch or sodium carboxymethyl-cellulose; and with lubricants such as talc or magnesium stearate. The active ingredient may also be administered by injection as a composition wherein, for example, saline, dextrose or water may be used as a suitable pharmaceutically acceptable carrier.
The dose when using the compounds and formulations described herein can vary within wide limits and as is customary and is known to the physician, it is to be tailored to the individual conditions in each individual case. It depends, for example, on the nature and severity of the illness to be treated, on the condition of the patient, on the compound employed or on whether an acute or chronic disease state is treated or prophylaxis is conducted or on whether further active compounds are administered in addition to the compounds. Representative doses include, but not limited to, about 0.001 mg to about 5000 mg, about 0.001 mg to about 2500 mg, about 0.001 mg to about 1000 mg, 0.001 mg to about 500 mg, 0.001 mg to about 250 mg, about 0.001 mg to 100 mg, about 0.001 mg to about 50 mg and about 0.001 mg to about 25 mg. Multiple doses may be administered during the day, especially when relatively large amounts are deemed to be needed, for example 2, 3 or 4 doses. Depending on the individual and as deemed appropriate from the patient's physician or caregiver it may be necessary to deviate upward or downward from the doses described herein.
The amount of active ingredient, or an active salt or derivative thereof, for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will ultimately be at the discretion of the attendant physician or clinician. In general, one skilled in the art understands how to extrapolate in vivo data obtained in a model system, typically an animal model, to another, such as a human. In some circumstances, these extrapolations may merely be based on the weight of the animal model in comparison to another, such as a mammal, preferably a human, however, more often, these extrapolations are not simply based on weights, but rather incorporate a variety of factors. Representative factors include the type, age, weight, sex, diet and medical condition of the patient, the severity of the disease, the route of administration, pharmacological considerations such as the activity, efficacy, pharmacokinetic and toxicology profiles of the particular compound employed, whether a drug delivery system is utilized, on whether an acute or chronic disease state is being treated or prophylaxis is conducted or on whether further active compounds are administered in addition to the compounds described herein and as part of a drug combination. The dosage regimen for treating a disease condition with the compounds and/or compositions is selected in accordance with a variety factors as cited above. Thus, the actual dosage regimen employed may vary widely and therefore may deviate from a preferred dosage regimen and one skilled in the art will recognize that dosage and dosage regimen outside these typical ranges can be tested and, where appropriate, may be used in the methods described herein.
The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations. The daily dose can be divided, especially when relatively large amounts are administered as deemed appropriate, into several, for example 2, 3 or 4 part administrations. If appropriate, depending on individual behavior, it may be necessary to deviate upward or downward from the daily dose indicated.
The compounds can be administrated in a wide variety of oral and parenteral dosage forms. It will be obvious to those skilled in the art that the following dosage forms may comprise, as the active component, either a compound described herein or a pharmaceutically acceptable salt, solvate or hydrate of a compound described herein.
For preparing pharmaceutical compositions, the selection of a suitable pharmaceutically acceptable carrier can be either solid, liquid or a mixture of both. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories and dispersible granules. A solid carrier can be one or more substances which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.
In powders, the carrier is a finely divided solid which is in a mixture with the finely divided active component.
In tablets, the active component is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted to the desire shape and size. The powders and tablets may contain varying percentage amounts of the active compound. A representative amount in a powder or tablet may contain from 0.5 to about 90 percent of the active compound; however, an artisan would know when amounts outside of this range are necessary. Suitable carriers for powders and tablets are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter and the like. The term “preparation” is intended to include the formulation of the active compound with encapsulating material as carrier providing a capsule in which the active component, with or without carriers, is surrounded by a carrier, which is thus in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets and lozenges can be used as solid forms suitable for oral administration.
For preparing suppositories, a low melting wax, such as an admixture of fatty acid glycerides or cocoa butter, is first melted and the active component is dispersed homogeneously therein, as by stirring. The molten homogenous mixture is then poured into convenient sized molds, allowed to cool and thereby to solidify.
Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or sprays containing in addition to the active ingredient such carriers as are known in the art to be appropriate.
Liquid form preparations include solutions, suspensions and emulsions, for example, water or water-propylene glycol solutions. For example, parenteral injection liquid preparations can be formulated as solutions in aqueous polyethylene glycol solution. Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed, as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.
The compounds according may thus be formulated for parenteral administration (e.g. by injection, for example bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative. The pharmaceutical compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g. sterile, pyrogen-free water, before use.
Aqueous formulations suitable for oral use can be prepared by dissolving or suspending the active component in water and adding suitable colorants, flavors, stabilizing and thickening agents, as desired.
Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, or other well-known suspending agents.
Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for oral administration. Such liquid forms include solutions, suspensions and emulsions. These preparations may contain, in addition to the active component, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents and the like.
For topical administration to the epidermis the compounds may be formulated as ointments, creams or lotions, or as a transdermal patch.
Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents.
Formulations suitable for topical administration in the mouth include lozenges comprising active agent in a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
Solutions or suspensions are applied directly to the nasal cavity by conventional means, for example with a dropper, pipette or spray. The formulations may be provided in single or multi-dose form. In the latter case of a dropper or pipette, this may be achieved by the patient administering an appropriate, predetermined volume of the solution or suspension. In the case of a spray, this may be achieved for example by means of a metering atomizing spray pump.
Administration to the respiratory tract may also be achieved by means of an aerosol formulation in which the active ingredient is provided in a pressurized pack with a suitable propellant. If the compounds or pharmaceutical compositions comprising them are administered as aerosols, for example as nasal aerosols or by inhalation, this can be carried out, for example, using a spray, a nebulizer, a pump nebulizer, an inhalation apparatus, a metered inhaler or a dry powder inhaler. Pharmaceutical forms for administration of the compounds as an aerosol can be prepared by processes well known to the person skilled in the art. For their preparation, for example, solutions or dispersions of the compounds in water, water/alcohol mixtures or suitable saline solutions can be employed using customary additives, for example benzyl alcohol or other suitable preservatives, absorption enhancers for increasing the bioavailability, solubilizers, dispersants and others and, if appropriate, customary propellants, for example include carbon dioxide, CFCs, such as, dichlorodifluoromethane, trichlorofluoromethane, or dichlorotetrafluoroethane; and the like. The aerosol may conveniently also contain a surfactant such as lecithin. The dose of drug may be controlled by provision of a metered valve.
In formulations intended for administration to the respiratory tract, including intranasal formulations, the compound will generally have a small particle size for example of the order of 50 microns or less. Such a particle size may be obtained by means known in the art, for example by micronization. When desired, formulations adapted to give sustained release of the active ingredient may be employed.
Alternatively the active ingredients may be provided in the form of a dry powder, for example, a powder mix of the compound in a suitable powder base such as lactose, starch, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidone (PVP). Conveniently the powder carrier will form a gel in the nasal cavity. The powder composition may be presented in unit dose form for example in capsules or cartridges of, e.g., gelatin, or blister packs from which the powder may be administered by means of an inhaler.
The pharmaceutical preparations are preferably in unit dosage forms. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.
Tablets or capsules for oral administration and liquids for intravenous administration are preferred compositions.
The compounds may optionally exist as pharmaceutically acceptable salts including pharmaceutically acceptable acid addition salts prepared from pharmaceutically acceptable nontoxic acids including inorganic and organic acids. Representative acids include, but are not limited to, acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethenesulfonic, dichloroacetic, formic, fumaric, gluconic, glutamic, hippuric, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, oxalic, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, oxalic, p-toluenesulfonic and the like. Certain pharmaceutically acceptable salts are listed in Berge, et al., Journal of Pharmaceutical Sciences, 66:1-19 (1977), incorporated herein by reference in its entirety.
Embodiments of the present disclosure provide compositions and methods for imaging. In some embodiments, the compositions are used as imaging agents in photoacoustic tomography (PAT). Photoacoustic (PA) detection relies on intrinsic absorption at specific excitation laser-generated wavelengths resulting in ultrasonic waves detected via conventional acoustic transducers. For imaging applications, longer wavelengths are advantageous because they afford greater imaging depth, with reduced potential for phototoxicity. Experiments conducted herein demonstrated that clofazimine yielded optimal PAT signals at 450 to 540 nm. Further experiments demonstrated that clofazimine accumulates in macrophages and thus provides a marker for inflammation.
Accordingly, in some embodiments, provided herein is the use of clofazimine or crystals comprising clofazimine as described herein as PAT imaging or contrast agents to identify inflammation. In some embodiments, PAT is used in combination with ultrasound imaging.
In some embodiments, PAT imaging with clofazimine contrast agents is utilizing to identify inflammation in a joint (e.g., a knee joint, a finger joint, a toe joint, a hip joint, and an elbow joint). In some embodiments, the inflammation is associated with arthritis in the joint. Thus, in some embodiments, the presence of a PAT signal associated with clofazimine in a joint is indicative of a diagnosis of arthritis or other inflammation in the joint.
In some embodiments, clofazimine and crystals thereof further find use in the treatment of inflammation in a joint (e.g., as described herein).
Mice (4 week old, male C57Bl6) were purchased from the Jackson Laboratory (Bar Harbor, Me.) and acclimatized for 1 week in a specific-pathogen-free animal facility. Animal care was provided by the University of Michigan's Unit for Laboratory Animal Medicine (ULAM) and the experimental protocol was approved by the Committee on Use and Care of Animals in accordance with NIH guidelines. An oral diet containing CFZ was fed to mice as previously described (Baik J, et al., Antimicrob. Agents Chemother. 2013; 57:1218-30; Baik J, et al., PLoS One 2012; 7:e47494; Keswani R K, et al., Mol. Pharm. 2015:10). CFZ (C8895; Sigma-Aldrich, St. Louis, Mo., USA) was dissolved in sesame oil (Roland, China, or Shirakiku, Japan) to achieve a concentration of 3 mg/ml, which was mixed with Powdered Lab Diet 5001 (PMI International, Inc., St. Louis, Mo., USA) to produce a 0.03% drug to powdered feed mix. A corresponding amount of sesame oil was mixed with chow for vehicle treatment (control mice). On average, food consumption for a 25 g mouse was 3 g per day, resulting in 10 mg of bioavailable drug/kg per day. For CFZ treatment, the drug diet was administered for 8 weeks followed by regular chow for 8 weeks (washout phase).
Isolation of CLDIs from Mouse Spleen.
At 8 weeks post drug feeding, mice were euthanized by exsanguination while deeply anesthetized by an intraperitoneal injection of ketamine (100 mg/kg)/xylazine (10 mg/kg) and spleens were harvested and cut open to prepare tissue homogenate in phosphate-buffered saline (PBS). The tissue homogenate was sonicated for 30 minutes and centrifuged (100×g for 1 minute) to remove large cell debris. A solution of 10% sucrose in PBS was added to the acquired supernatant and the mixture was centrifuged (100×g). The resulting supernatant was centrifuged (21,000×g for 1 min) to pellet drug inclusions which were then resuspended in 2 ml of 10% sucrose. CLDIs were further purified using a 3-layer discontinuous gradient (50%, 30% and 10% sucrose in PBS) centrifugation method (3200×g for 30 min, no brakes) (Yoon G S, et al., Mol. Pharm. 2015:10). The CFZ content of the isolated CLDIs was determined spectrophotometrically (λ=495 nm) by procuring 100 μl of CLDIs (in triplicate) by centrifugation (21,000×g for 1 min) and dissolution in DMSO followed by comparison with calibrated CFZ standards.
CFZ was dissolved in DMSO to achieve a concentration of 20 μM. Fluorescence excitation and emission scans were done in increments of 10 nm from 400 nm to 800 nm on a Perkin-Elmer LS-55 fluorescence spectrometer using standard cuvettes. Data were imported into Microsoft® Excel (Redmond, Wash., USA) (MS-Excel) for further analysis. The fluorescence yield was background-subtracted using data obtained from solvent alone (DMSO) and was normalized to the maximum fluorescence yield measured across the spectral wavelength range tested.
For preparation of slides, CFZ drug crystals were dusted on a glass slide followed by the application of a glass cover slip. For slides of CLDIs, a 20 μl drop of purified CLDIs was placed on a glass slide and allowed to dry overnight in the dark. The following day, a single drop of Prolong® Gold (Life Technologies, Carlsbad, Calif.) was added to the CLDIs and a cover slip was applied prior to imaging. Spectral confocal microscopy was performed on a Leica Inverted SP5X confocal microscope system with 2-photon FLIM (Leica Microsystems Inc., Buffalo Grove, Ill.) using excitation wavelengths (λ=470-670 nm). Image analysis and quantification was performed on Leica LAS AF. Several regions of interest (ROIs) of individual crystals were used to obtain fluorescence data which were imported into MS-Excel for further analysis. All fluorescence yields were normalized to the maximum fluorescence yield measured across the spectral range tested and background subtracted using data obtained from a blank slide.
Visualization of all samples (cells or crystals) was done on a Nikon Eclipse Ti (Nikon Instruments Inc., Melville, N.Y., USA). The fluorescence filters (Excitation/Emission) used were DAPI (350/405 nm, exposure=50 ms), FITC (490/510 nm, exposure=100-500 ms), Texas Red (590/610 nm, exposure<500 ms) and Cy5 (640/670 nm, exposure=15 ms). Brightfield color photographs were acquired using a Nikon DS-Fi2 camera while fluorescence photographs were acquired using a Photometrics® CoolSNAP™ MYO (Photometrics, Tucson, Ariz., USA) camera. CLDIs (seen as intense red pigmentation) were counted and analyzed for physical dimensions using the Nikon Elements software (Nikon Instruments Inc., Melville, N.Y., USA).
Macrophages phagocytose CLDIs isolated from mouse spleen following 8 weeks of CFZ treatment (14). RAW 264.7 cells (TIB-71™ ATCC, Manassas, Va.) cells were maintained with DMEM+10% fetal bovine serum (FBS) (10082; Gibco®, Invitrogen, Carlsbad, Calif., USA) with 1% penicillin/streptomycin (15140; Gibco®, Invitrogen, Carlsbad, Calif., USA) at 37° C., 5% CO2. The cells were seeded at 4×105 cells/well in a 6-well plate 18-20 hours prior to incubation with isolated and purified spleen CLDIs at a solution equivalent concentration of 40 μM CLDIs (14). Following 24 hours post CLDI incubation, cells were gently scraped and suspended in sterile flow cytometry tubes at a density of ˜2×106 cells/ml of phosphate-buffered saline (PBS)+5% FBS. The cells were analyzed on a MoFlo® Astrios™ (Beckman Coulter, Brea, Calif., USA) using various laser combinations. Unless otherwise mentioned, forward and side scatter were measured using the 488 nm laser. Laser settings are referenced in the following format—Excitation Emission/Bandwidth in nm. For example, if the excitation laser used is 488 nm and the emission detector is at 576 nm with a bandwidth of 21 nm, the written format is 488 576/21. To check the activity of viability dyes and signal compensation, just prior to analysis, propidium iodide (PI) (00-6990-50; Affymetrix eBioscience, San Diego, Calif., USA) or 4,6-diamidino-2-phenylindole, dihydrochloride (DAPI) (D1306; Life Technologies, Grand Island, N.Y., USA) was added to the cells (5 μl per 1×106 cells). All gating and analysis was done on at least 10,000 cells (DAPI(−) or PI(−) cell population for viability studies) using FlowJo (FlowJo, LLC, Ashland, Oreg., USA). Statistical analysis of sensitivity and specificity was conducted by acquiring >6 brightfield microscopy images of sorted cell populations on standard microscopy slides and >350 cells were counted for each sorting experiment. Sensitivity and specificity were calculated as follows.
Where TP (True Positive)=CLDI(+) in CLDI(+) cell population,
FP (False Positive)=CLDI(−) in CLDI(+) cell population,
TN (True Negative)=CLDI(−) in CLDI(−) cell population,
FN (False Negative)=CLDI(+) in CLDI(−) cell population,
Peritoneal lavage was done as previously reported before and after the initiation of CFZ or vehicle treatment (1 week, 2 weeks, 4 weeks, 8 weeks and 16 weeks (8 weeks drug feed+8 weeks washout phase)) (16). Mice were euthanized as described above followed by sterilization of the outer skin with 70% ethanol. A small incision was made along the midline of the abdomen followed by abdominal skin retraction up to the thoracic boundary and the animal extremities to expose the intact peritoneal wall. A smaller incision was then made on the peritoneal wall to expose the cavity. The entire peritoneal cavity was washed with ice-cold sterile PBS+5% FBS (5-10 ml) and collected as peritoneal exudate. The exudate was then centrifuged (100×g for 5 min, 4° C.) and resuspended in 1.5 ml of PBS+5% FBS. Cells were counted using a hemocytometer for viable cells using Trypan Blue and for CLDI-containing cells.
Mice were euthanized as described above and the trachea was surgically exposed and cannulated with an 18G luer stub and the lungs were lavaged to obtain alveolar exudate by instilling calcium- and magnesium-free Dulbecco's PBS (DPBS) containing 0.5 mM EDTA in 1-ml aliquots for a total of 6 ml. The alveolar exudate fluid was centrifuged (400×g, 10 min, 4° C.) and resuspended in RPMI 1640 media. Viable (using Trypan Blue staining method) and CLDI containing cells were counted using a hemocytometer.
Peritoneal and alveolar lavage was performed as stated before to obtain the respective exudates. Purified Fc block CD16/32 (1 μl) was added for every 100,000 cells for analysis. For CLDI fluorescence signal experiments, cells were pelleted by centrifugation (100×g for 5 min) followed by resuspension in PBS+5% FBS (500 μl). For functional assays, antibodies—anti-F4/80-eFluor 450 (48-4801, Affymetrix eBioscience, San Diego, Calif., USA), anti-CD86-FITC (553691, BD Biosciences, San Jose, Calif., USA), anti-CD206-FITC (141704, BioLegend®, San Diego, Calif., USA), anti-CD11c-eFluor450 (48-0114, Affymetrix eBioscience) and anti-Ly6G-eFluor450 (48-5931, Affymetrix eBioscience) were added to the cell suspension at a loading of 1 μg/100,000 cells (1:10 volumetric ratio) and incubated in the dark (30 min, 4° C.). Following incubation, the samples were diluted with PBS and pelleted by centrifugation (100×g, 5 min). The supernatant was discarded and the pellet was re-suspended in 300 μl of PBS+2% FBS. Just prior to analysis, PI (5 μl per 1×106 cells) was added to the cells to assess viability. Sample measurements were done on a MoFlo® Astrios™ EQ. All gating and analysis was done on at least 10,000 live cells (PI(−) cell population) using FlowJo. CLDI(+) cells were gated either using 640 671/30 or 640 795/70. A small sub-population of CD206(+) cells, which was present in both CLDI(+) F4/80(−) and CLDI(−) F4/80(+) populations, exhibited high CD206 expression based on the intensity of the fluorescence signal. These cells also had extremely high side-scatter that were measured as saturated signals. This small sub-population of cells were not considered for evaluation of change of CD206 expression in the peritoneal macrophages.
CLDI treated RAW264.7 cells were generated as mentioned above but on cover-slips in a 6-well plate. The cells were washed with PBS buffer following which Hoechst 33342 (Invitrogen, Carlsbad, Calif.) and FM® 1-43 (Molecular Probes T35356, Invitrogen) were used to stain cell nuclei and the plasma membrane, respectively. Cells were incubated with 1:1 (v/v) dye mixtures of 5 μg/ml Hoechst 33342 and 7 μM FM® 1-43 in HBSS (300 μl) for 15 min at room temperature. The confocal imaging of the live cells was performed on an Olympus Fluoview 500 (Olympus America Inc., Center Valley, Pa., USA) using lasers for DAPI (405 450/50), FITC (488 525/50) and Cy5 (640 671/30) channels. Z-stack images of the cells were captured along the Z-axis (interval=0.25 μm) and analyzed using the Nikon NIS-Elements 3.2 confocal software (Nikon Instruments Inc., Melville, N.Y.). For peritoneal macrophages stained with F4/80, a drop of the stained cell sample was mounted on a blank microscopy slide; Prolong Gold and a cover-slip were applied followed by immediate scanning.
Plots were constructed using Origin 9.0 (OriginLab Corporation, Northampton, Mass., USA) and laid out in figure format using scalable vector graphics format (svg) in either Inkscape or GIMP. Flow cytometry plots were obtained in svg format directly from FlowJo and assembled using Inkscape. All statistical analysis was performed using Student's t-test in MS-Excel. Correlation statistics were done using a Pearson's test in Origin 9.0. Results were considered significant if p≦0.05. MIFlowCyt compatible information was prepared using established standards Cytom. Part A 2008; 73:926-930).
The fluorescence excitation and emission scan of CFZ dissolved in DMSO indicates that CFZ in solution is fluorescent in the range—Excitation: 540-560 nm, Emission: 560-600 nm (
Identification of CLDI-Containing Cell Subpopulations with Standard Flow Cytometer Configurations.
To design a single-cell analysis technique to study the pharmacology of xenobiotic-sequestering macrophages and the impact of CLDIs on cellular functions, it was determined whether flow cytometery could be used to distinguish between cells that contain and do not contain CLDIs. Samples were analyzed using standard excitation lasers and detector configurations, to determine the specific fluorescence cytometry settings that can be used for analyzing CLDI containing cell subpopulations and to establish any spectral overlap detected in the other channels (
To confirm these observations, a fluorescence activated cells sorting experiment was performed using 488 664/22, 561 692/75 and 640 671/30 along with laser-scanning confocal microscopy of incubated cells. When RAW264.7 cells incubated with CLDIs and labeled with fluorescent cellular staining dyes—Hoechst 33342 (nucleus) and FM 1-43 (plasma membrane) were imaged, the intracellular accumulation of CLDIs was confirmed via their far-red fluorescence (
In flow cytometry experiments, a fundamental functional assay involves discriminating live and dead cells using standard fluorescent labels. Accordingly, to determine whether CLDI-containing cells were viable, RAW264.7 cells loaded with CLDIs were further incubated with membrane impermeant cell viability dyes—PI or DAPI prior to flow cytometric analysis. Using the laser-detector settings for PI—561 614/20, two populations were detected using CLDI-incubated RAW264.7 cells. A compensation of the spectral overlap that was observed at 561 614/20 was conducted to verify that PI can be used in the presence of CLDIs (at 640 671/30) without signal overlap (Figure B). Incubating control (CLDI-free) cells with PI yielded two distinct cell populations, corresponding to live (low fluorescence signal) and dead (high fluorescence signal) cells (
In Vivo Flow Cytometry Detection of CLDI-Containing Cells in Peritoneal and Alveolar Exudates from CFZ-Treated Mice
To measure the presence of CLDIs in peritoneal exudates obtained from CFZ-treated mice, a quantitative, flow cytometric analysis was performed on peritoneal exudate of mice following 8-week drug administration. While the control mouse peritoneal exudate showed two distinct populations (labeled 1 and 2—
Next, it was determined whether the time course of CLDI accumulation in macrophages as determined by flow cytometry corresponded to the expected time course of CLDI accumulation following drug treatment (Baik, 2013, supra; Baik 2012, supra). Following drug treatment of mice for varying time periods, peritoneal exudates were obtained and analyzed by flow cytometry as well as light microscopy, revealing that CLDIs were not present at 1 week and 2 weeks, but could be detected starting at 4 weeks (
To determine whether flow cytometry could be used to characterize the molecular phenotype of CLDI-containing macrophage sub-populations, multi-stain flow cytometry was conducted on peritoneal exudates of CFZ-treated animals. For this purpose, cells obtained from peritoneal exudates were incubated with other commonly employed macrophage-targeted antibodies possessing fluorescent signals distinct from that of CLDIs (405 448/59 and 488 513/26;
To follow up on the analysis on peritoneal exudate, a flow cytometric analysis was performed on alveolar exudate of drug fed mice at 8 weeks to confirm the macrophage-specific accumulation of CLDIs in the lung (Baik et al., 2012, supra; Conalty M L, Jina A G. The Antileprosy Agent Clofazimine (B.663) in Macrophages: Light, Electron Microscopy and Function Studies. In: Reticuloendothel. Syst. Immune Phenom. Springer; 1971. p 323-331; Conalty M L, et al., Br. J. Exp. Pathol. 1962; 43:650-654) (
Correlation of CLDI Amount with Mean Cellular Fluorescence Intensity (MFI)
In fluorescence activated cell sorting experiments, attempts to relate the MFI of sorted cell populations with the total number of CLDIs per cell did not yield interpretable data. Therefore, to determine whether the fluorescence signal per cell was related to the number of CLDIs in each cell, RAW264.7 macrophage cells were incubated with CLDIs, and fluorescence images were acquired with conventional epifluorescence microscopy using the Cy5 filter for CLDIs (
In summary, the analysis of macrophage populations in vitro and ex vivo was made possible due to the unique fluorescence spectral characteristics of CLDIs relative to neutral free base CFZ precipitates as well as the CFZ molecule in soluble form. The specific fluorescence of CLDIs was utilized in single-cell flow cytometry to study macrophage-related intracellular drug accumulation wherein it was possible to measure macrophage functions including viability and expression of different cell surface markers, by exploiting the unique fluorescence spectrum of CLDIs and the availability of many other orthogonal fluorescent probes that can be detected with standard flow cytometry parameters, with minimal spectral overlap from the CLDI signal. It was also confirmed that the presence of CLDIs has a minor polarizing effect towards the M2 macrophage phenotype. More importantly, the unique fluorescence shift of CFZ molecules from the freely soluble green-blue fluorescent state to the solid far-red CLDI fluorescent state facilitates analysis of the molecular mechanisms driving drug accumulation and CLDI formation, using a variety of molecular, pharmacological, genetic, or systems biology approaches. These studies are useful for furthering understanding of the role of xenobiotic sequestering macrophages in the response of the organism to bio-accumulating drugs or environmental toxicants.
The initial screening to obtain a short-list of drugs from Drugbank was done via Boolean search to extract all small molecule drugs that were FDA-approved, not withdrawn nor illicit and containing the descriptive term “anti-inflammatory” resulting in a short list of 126 molecules. Two 1536-well plates containing a common subset of the NCGC Pharmaceutical Collection (NPC) (Huang, R. et al. Sci. Transl. Med. 3, 80ps16 (2011)) were selected to be analyzed for visible color. The plates included 87 short-listed drugs solubilized in dimethyl sulfoxide (DMSO) up to a concentration of 20 mM that were then photographed using a flatbed scanner and measured for their UV/Vis absorbance profiles. The raw PNG formatted image was manually cropped and stored as 2 separate PNG images. A simple custom Java program was then used to select the centremost pixel from each well and report the colour information in both RGB and HSV format. Molecules not yet available in NPC format (39 in number) were searched in literature, USP Pharmacopeia and other databases for their colour and absorption spectra. To re-evaluate the UV/Vis profile of the 10 pigmented compounds, samples were prepared at a concentration of 0.2-1 mM in DMSO and spectra was recorded from 300-1000 nm on a Biotek II multi-well plate reader. The solution absorbance was normalised with the corresponding value using a control sample containing DMSO alone and plotted from 300 to 600 nm as no discernible absorbance was measured for any of these compounds at >600 nm.
The setup for PA spectroscopy measurement is shown in
p(λ)=ΓΦμa(λ)
where Γ is the Gruneisen parameter, which is a constant and Φ is the laser fluence, which was calibrated using the reference black body (Feng, T. et al. Ting. Opt. Lett. 40, 1721-1724 (2015)). Clofazimine (CFZ) as a free base was obtained directly from Sigma-Aldrich (St. Louis, Mo., Catalogue No. C8895) and solubilised in DMSO and confirmed via 1H-NMR to be unprotonated in solution (Keswani, R. K. et al. Mol. Pharm. 12, 2528-2536 (2015)). To obtain CFZ-H+Cl− in solution, CFZ-HCl crystals, obtained via crystallisation at pH 5 as published before (Keswani, R. K. et al. Mol. Pharm. 12, 2528-2536 (2015), were resolubilized in DMSO. Briefly, equal volumes of 1 M NH4Cl in H2O and 2 mM CFZ in methanol were mixed with the addition of a surfactant (up to 2% (v/v) Triton™ X-100, Sigma-Aldrich, Catalogue No. X100) and incubated at room temperature for 48 hours. The precipitates obtained were washed twice with H2O and lyophilized followed by storage at −20° C. until used. Just prior to analysis, CFZ-H+Cl− crystals were solubilized in DMSO. These solubilized samples were also confirmed to be monoprotonated via 1H-NMR. For diprotonated CFZ, free-based CFZ crystals were dissolved in 9 M H2SO4 resulting in the formation of a deep purple coloured solution of diprotonated CFZ.
Experiments using the CFZ mice model were performed as published before (Baik et al., 2013, supra; Baik et al., 2012, supra; Keswani, R. K. et al. Mol. Pharm. 12, 2528-2536 (2015)). Mice (4 week old, male C57Bl/6) were purchased from the Jackson Laboratory (Bar Harbor, Me.) and acclimatised for 2 weeks in a specific-pathogen-free animal facility. All animal care was provided by the University of Michigan's Unit for Laboratory Animal Medicine (ULAM), and the experimental protocol was approved by the Committee on Use and Care of Animals. CFZ was dissolved in sesame oil (Roland, China, or Shirakiku, Japan) to achieve a concentration of 3 mg/ml, which was mixed with Powdered Lab Diet 5001 (PMI International, Inc., St. Louis, Mo.) to produce a 0.03% drug to powdered feed mix. A corresponding amount of sesame oil was mixed with chow for vehicle treatment (control).
The murine MΦ cell line RAW264.7 was purchased from ATCC (Manassas, Va.) and maintained in growth media (DMEM (Life Technologies, Carlsbad, Calif.) supplemented with 10% FBS and 1% penicillin/streptomycin). Isolated CLDIs were added at 20 μM solution equivalent concentration in growth media to 8-well chamber-slides (Nunc™ Labtek™ II, Thermo-Scientific Catalogue No. 154534) containing 5×104 RAW264.7 cells/well and incubated (24 h at 37° C. and 5% CO2). Following incubation, cells were fixed using 4% paraformaldehyde in PBS for 15 mins at room temperature followed by rinsing and washing with ice-cold PBS twice. The samples were counterstained with Hoechst33342 for nuclear labelling followed by removal of chambers. A drop of Prolong Gold® (Life Technologies) was placed on the sample and covered with a glass coverslip for imaging.
The setup for PA microscopy measurement as described previously is shown in
where μs and μb are the means of the signals in the region of interest (ROI) and the background respectively and σs and σb are the corresponding standard deviations. Signal intensity histograms of the acquired images were constructed via measurements in ImageJ (National Institutes of Health, Bethesda, Md.)
Mice Carrageenan Footpad Edema Model. Carrageenan (C1867, Sigma-Aldrich®, 2% in PBS, 30 μl) was injected into the right footpad while PBS (30 μl) was injected in the left footpad as a negative control. For preliminary testing of CFZ as an anti-inflammatory agent, 8-week orally fed mice (CFZ and control) were used. For testing of CFZ in soluble and microcrystal form, naïve 4-5 week old mice were injected with carrageenan first followed by injection of CFZ-A (2 mM in DMSO, 30 μl), CLDIs (2 mM equivalent in PBS, 30 μl) or CFZ-HCl (2 mM equivalent in PBS, 30 μl) at 48 h post carrageenan injection. For control experiments, CFZ-A, CLDIs and CFZ-HCl were injected into the right footpads of naïve 4-5 week old mice. DMSO was used as a control for CFZ-A in the left footpad whereas PBS was used as a control for CLDIs and CFZ-HCl. Footpads were measured (length—l, width—w, three thicknesses—t1, t2, t3) at various time points using a digital vernier calliper and changes in the swelling based on volume of the foot was calculated as per above.
8% gelatin from porcine skin (G2500, Sigma) was dissolved in water, and then heated and stirred on a hotplate for about one hour. After cooling down, several wells were dug out of the surface and filled with CFZ samples. After that, smaller 8% gelatin phantoms was used to cover the surface of the previous one to seal the embedded samples.
A cadaver hand was requested from Anatomical Donation Program at University of Michigan through an organ donation program. Once received, the cadaver tissue was frozen in a lab specimen freezer under −20° C. Before the imaging experiment, it was submerged in flowing cold water and thawed for about one hour. The request, transportation, storage, and handling of the cadaver tissue followed the policies and guidelines of Michigan Anatomical Gift Law Public Act 368 of 1978, amended as Public Act 39 of 2008. After the experiment, the cadaver tissue was returned to the Anatomical Donation Program.
The details of the hardware used have been reported in previous publications (Xu, G. et al. J. Biomed. Opt. 18, 010502 (2013); Yuan, J. et al. J. Biomed. Opt. 18, 86001 (2013)) and a schematic and setup is shown in
For the clinical human model imaging, a 23 G syringe needle with a nominal outer diameter of 0.64 mm was inserted into the target metacarpophalangeal (MCP) joint of the cadaver hand transcutaneously, guided by the US-PA dual-modality imaging system with its tip stopped next to the joint about 5.6 mm beneath the skin surface. A commercial ultrasound machine (z.one ZONARE) equipped with a linear probe (L10-5, ZONARE) was used to further confirm the positioning of the needle in the joint. CFZ-A at 5 mM in DMSO solution was injected slowly into the joint via the inserted syringe needle. The ROI and background in the reconstructed PA images were segmented and colored based on different color schemes for the purpose of illustration. The laser fluence on the skin surface was estimated to be 4 mJ/cm2, which is well below the American National Standards Institute (ANSI) safety limit at 532 nm. The intensities of both the phantom images and the human model images were normalized.
Photoacoustic (PA) detection relies on intrinsic absorption at specific excitation laser-generated wavelengths resulting in ultrasonic waves detected via conventional acoustic transducers (Wang, X., et al., Biomed. Opt. Express 1, 1117-1126 (2010)). For imaging applications, longer wavelengths are advantageous because they afford greater imaging depth, with reduced potential for phototoxicity (Ntziachristos, V. Nat. Methods 7, 603-14 (2010)). Therefore, to search for candidate small molecule drugs with potential applications as PA contrast agents, the optical properties of FDA-approved anti-inflammatory drugs were assayed. Every organic molecule has a characteristic optical absorption spectrum with colored or pigmented compounds absorbing more light at higher wavelengths relative to colorless compounds (Lewis, G. N. & Calvin, M. Chem. Rev. 25, 273-328 (1939); Sklar, A. L. J. Chem. Phys. 5, 669-681 (1937)). After identifying drugs with the strongest optical absorbance and PA signals at visible wavelengths, PA-specific hardware was used to detect the drug in a variety of experimental and clinically-relevant platforms and tested their MΦ-targeting and anti-inflammatory capabilities in a relevant animal model.
Currently, there are 1602 FDA-approved small molecule drugs on the market catalogued in the Drug Bank (Law, V. et al. Nucleic Acids Res. 42, 1091-1097 (2014)). These drugs were sorted to focus on those with clinically established anti-inflammatory activity (126 in number). In order to screen them for intrinsic color with an optical absorption spectrum in the visible color range or near IR range, these short-listed drug molecules were assayed using a combination of optical photography, color identification, literature survey and high-throughput absorption spectrophotometry. While the majority of the compounds were colorless, eight drugs were selected for further testing based on their optical properties while two other molecules were identified to be colored via a parallel literature search. Four of these 10 drugs showed high absorbance at wavelengths (λ)>400 nm (
A PA spectroscopy unit was used to quantitatively establish the spectral properties of CFZ's PA signal (
Many animal models to study the clinical pharmacology and pharmacokinetic properties of CFZ have been previously established (Cholo et al., 2012, supra; Banerjee, D. K. et al. Am. J. Trop. Med. Hyg. 23, 1110-1115 (1974)). Independently, to confirm the MΦ-associated accumulation of CFZ, immunofluorescence analyses were conducted using a positive MΦ marker (CD68) (Zhang, X., et al., Curr. Protoc. Immunol. 9, 1-18 (2008)) on histological sections of the spleen and liver from 8 week orally fed CFZ mice. Bright red pigmented spots, consistent with the natural color of the drug, were observed in both the organs (
These fluorescent red inclusions co-localised with CD68 expression in both the spleen and liver (
To quantitatively confirm and establish the PA signal intensity of these MΦ-containing CFZ inclusions, spleen and liver sections of CFZ-treated mice were imaged using a PA microscope using optical excitation at 532 nm (
To target the translation from bench to bedside, sufficient imaging depth beneath the sample surface is usually desired. Photoacoustic Tomography (PAT) could offer much better imaging depth beyond the optical mean free path. To establish the suitability of CFZ for PAT, gelatin phantoms were prepared, embedded with sample wells containing soluble CFZ as free-base or in monoprotonated form in DMSO (resolubilized CFZ-H+Cl−, hereby referenced as CFZ-A), CFZ inclusions isolated from the spleen of drug-treated mice (hereby referred to as Crystal-like-drug-inclusions or CLDIs) in PBS and their synthetic crystal analogue—CFZ-HCl microcrystalline particles in PBS (Keswani, R. K. et al. Mol. Pharm. 12, 2528-2536 (2015)) (
To confirm the in vivo anti-inflammatory activity of PA detectable forms of CFZ, an established carrageenan-based footpad-edema model (Posadas, I. et al. Br. J. Pharmacol. 142, 331-8 (2004)) (swelling measurement model shown in
A clinical imaging platform, combining dual modality imaging via PAT and ultrasound (US) (Xu, G. et al. J. Biomed. Opt. 18, 010502 (2013)) (US-PA) was established to determine whether CFZ is useful as a contrast agent for the diagnosis or treatment of arthritis (
All primary antibodies were purchased from Cell Signaling Technology (Danvers, Mass.), except p65 and TLR4 (Abcam, UK), actin and TLR2 (Sigma, St. Louis, Mo.), and TLR9 (Thermo Pierce, Rockford, Ill.). Pam3 and LPS were purchased from Invivogen (San Diego, Calif.).
Purification of CFZ Crystal-Like Drug Inclusions (CLDIs) from Mice Spleen and CFZ Quantification
Clofazimine (Sigma-Aldrich, C8895) was prepared in sesame oil (Shirakiku, Japan, or Roland, China) and Powdered Lab Diet 5001 (PMI International, Inc., St. Louis, Mo.) and orally administrated to C57BL6 mice (4 week old, Jackson Laboratory, Bar Harbor, Me.) for 8 weeks as previously described (Baik et al, 2012, supra; Baik et al., 2013, supra).
Spleens were harvested and CLDIs were isolated using a previously described method with some modifications (Baik et al., 2011, supra). The spleens were cut into small pieces, homogenized with a syringe plunger and then filtered through a 40 μm cell strainer to remove connective tissue debris. The spleen homogenate was centrifuged (300×g for 10 min) to remove large cell debris and the pelleted CLDIs were resuspended in 10% sucrose in Dulbecco's PBS (DPBS) without CaCl2 and MgCl2, pH 7.4. CLDIs were further purified using a 3-layer discontinuous gradient (50%, 30% and 10% sucrose in DPBS) centrifugation method (3200×g for 30 min). For incubation with RAW 264.7 cells, CLDIs were washed 3 times with DPBS to remove sucrose and resuspended in DMEM with 5% FBS. Protein concentration of purified CLDI isolates before and after gradient centrifugation and subsequent washing with DPBS was determined using the bicinchoninic acid detection (BCA) assay (Thermo Pierce). CFZ content was spectrophotometrically measured using a previously described method with some modifications (Baik, J. et al., Mol. Pharm. 2011, 8, 1742-1749; Baik et al, 2013, supra). CLDIs in DPBS (100 μl of sample) were mixed with equal volume of xylene to create lipid-aqueous partitioning and then vortexed to dissolve CLDIs and extract the CFZ into the organic phase. The CFZ-containing xylene was placed into a new tube. Fresh xylene was added to the sample and the process was repeated twice until there was no CFZ remaining in the aqueous phase. The CFZ content in xylene was extracted twice using equal volume of 2.5 M H2SO4 and vortexing until there was no CFZ remaining in xylene. Final CFZ concentration was calculated from the standard curve generated by adding a known amount of drug solution to 2.5 M H2SO4 and measurement of absorbance at 530 nm (Synergy-2 plate reader; Biotek, Winooski, Vt.). The average extraction yield of CFZ was 90%, with elimination of 99% of protein.
Culture of RAW 264.7 Cells with Soluble CFZ or CLDIs and TLR Stimulation
The murine macrophage cell line RAW 264.7 was purchased from ATCC (Manassas, Va.) and maintained in DMEM (Life Technologies, Carlsbad, Calif.) supplemented with 10% FBS. Soluble CFZ or isolated CLDIs were added at various concentrations in DMEM with 5% FBS to 6-well plates containing 4×105 cells/well and incubated (24 h at 37° C. and 5% CO2). Culture supernatants were harvested at 24 h, centrifuged (1500×g for 5 min), and stored (−20° C.) in frozen aliquots prior to analysis. For experiments involving TLR stimulation, cells were washed twice with pre-warmed DPBS to remove extracellular, non-phagocytosed CLDIs or CFZ, and then the cells were serum-starved for 18 h in DMEM. Supernatants were collected from unstimulated cells and cells stimulated with 200 ng/ml Pam3 or 1 μg/ml LPS in DMEM.
The ability of RAW 264.7 cells to phagocytose CLDIs was measured by incubating cells with increasing concentrations of CLDIs for 24 h, as described above. The cells were then washed twice with pre-warmed PBS to remove extracellular CLDIs and images were captured using the Nikon Eclipse Ti (Japan) inverted microscope with brightfield to count cells and fluorescence at Cy5 wavelength to count CLDIs. Cells and CLDIs from each image were manually counted to calculate the percentage of CLDI-containing cells and the mean number of CLDIs internalized by each cell. A minimum of 5 random images were analyzed for each concentration (minimum 640 total cells counted). CLDI internalization was confirmed by confocal microscopy, following labeling of the plasma membrane of cells with the lipophilic, fluorescent styryl probe FM-143.
Cells were cultured with CFZ or CLDI media for 24 h in chamber slides (Lab Tek) and stained with 150 nM of MitoTracker Red CMXRos (MTR, Life Technologies) in fresh media for 45 min, and then NucBlue Live Cell Stain (Life Technologies) was applied for 10 min for nuclear staining. After incubation, extracellular MTR and NucBlue were washed twice with DPBS and the cells were visualized on a Nikon Eclipse Ti fluorescence microscope (Japan) using Texas Red (Mitotracker) and DAPI (NucBlue) filters. Real-time live cell images were captured every 10 sec for 30 min with the same microscope and video was created using the Nikon Elements software. For confocal imaging of intracellular CLDIs, FM 1-43 (Life Technologies) was used at 3.5 μM (15 min at 37° C.) for membrane staining followed by NucBlue (10 min). After washing with DPBS, cells were visualized using laser-scanning confocal microscopy (Olympus Fluoview 500) fitted with argon (FITC) and HeNe Red lasers (Cy5). Images were taken along the z-axis with a 60× objective at 0.25 μm intervals and the composite Z-stack images were created using the Nikon Elements software.
Brightfield and fluorescence (DAPI, FITC, Texas Red and Cy5) images were captured using the Nikon Eclipse Ti (Japan) inverted microscope equipped with a Nikon Digital Sight DS-Fi2 camera (Japan) for brightfield and Photometrics Coolsnap Myo camera (Tucson, Ariz.) for fluorescence. Polarized images were acquired with CRi Abrio Imaging System (Hinds Instruments, Hillsboro, Oreg.) fitted on the same microscope with a 623 nm polarizing filter using the OpenPolScope plugin for ImageJ and Micro-Manager (Mehta, S. B. et al., J. Opt. 2013, 15). For determining the ratio of nucleus-to-cytoplasm p65 fluorescence, ImageJ was used following previously described methods (Fuseler, et al., Microsc. Microanal. 2006, 12, 269-276; Noursadeghi, M. et al., J. Immunol. Methods 2008, 329, 194-200). The average volume of CLDIs was calculated using the area, Feret max and min values of each CLDI acquired from ImageJ. Each CLDI was considered to be cylindrical in shape. A detailed diagram of the calculation method is shown in
Cells were plated in triplicate wells at a density of 5×103 per well in 96-well plates in DMEM with 5% FBS and allowed to adhere overnight. Soluble CFZ (Stock solution 5 mM in DMSO) or CLDIs were added (0.25, 0.5, 1, 2, 4, 10, 20, 40 and 80 μM final concentrations) to cells and incubated (37° C.) for 24 h. XTT assay (Roche, UK) was carried out according to the manufacturer's instructions with absorbance measured at 450 nm and 690 nm using a Synergy-2 plate reader (Biotek). The cell viability percentage was calculated by comparing absorbance of CFZ and CLDI-treated cells to control (untreated) cells.
The media of cells with or without 6 h TLR stimulation by Pam3 or LPS was harvested, and TNFα and IL-1RA levels were measured by ELISA (Duoset, R&D Systems, Minneapolis, Minn.) in duplicate wells according to the manufacturer's instructions. The cytokine concentrations were expressed as picogram per milligram of cell lysate. Experiments were repeated three times and values are expressed as the mean±SD.
CFZ- or control chow-fed mice were euthanized by exsanguination while deeply anesthetized by intraperitoneal injection of 300 μl ketamine/xylazine. The trachea was surgically exposed and cannulated with an 18 G needle and the lungs were lavaged by instilling DPBS containing 0.5 mM EDTA in 1 ml aliquots for a total of 6 ml. Approximately 90% of the bronchoalveolar lavage (BAL) was retrieved. BAL was then centrifuged for 10 min at 400×g, 4° C. and resuspended in RPMI 1640 media (Life Technologies). The cells were placed in 12-well culture plate (Corning, Tewksbury, Mass.) and washed with media after 45 min, enabling the isolation of alveolar macrophages by adherence. The cells were imaged in brightfield and lysed in RIPA buffer (Sigma) for Western blot.
All data were expressed as mean±standard deviation. Statistical analysis was performed with one-way analysis of variance (ANOVA) and Bonferroni's post-hoc comparisons, or with Student's t-test (paired, two-way). Correlation analyses were performed using a Pearson's correlation coefficient measurement. All statistical analyses employed the IBM SPSS software and p≦0.05 was considered statistically significant.
Clofazimine CLDI Purification from Spleen.
In order to obtain pure CLDIs, a method to isolate and purify CLDIs from the spleen of 8 wk CFZ-fed mice using a 3-layer discontinuous gradient centrifugation method was utilized. Brightfield, polarized and fluorescence microscopy images (
Comparing the optical properties of CLDIs before and after the purification process thus showed that CLDIs were not altered by the purification process. The size dimensions of spleen CLDIs displayed as rod-shaped crystals (
The red color of CFZ allowed monitoring the cellular uptake of soluble CFZ or CLDIs (
CLDIs induced minimal cytotoxicity (
Changes in mitochondrial membrane permeability such as those leading to difference in MTR staining are upstream of activation of apoptosis pathways mediated by activation of caspases, proteolysis of caspase substrates, and ultimately leading to cell death. Since previous studies also indicated that soluble CFZ could activate apoptosis pathways in cultured macrophages (Magenau, A. et al., Traffic 2011, 12, 1730-1743), experiments were conducted to directly establish the activation of apoptotic pathways by monitoring caspase-3 and PARP cleavage after CFZ or CLDI treatment. Indeed, while soluble CFZ caused increased PARP cleavage at low concentrations (4 and 10 μM;
Previous studies on mice fed with a CFZ-supplemented diet for an 8 week period indicated increased levels of interleukin 1 receptor antagonist (IL-1RA) an endogenous, secreted anti-inflammatory signaling molecule (Baik et al., 2013, supra). Thus, it was established whether CFZ or CLDIs boosted IL-1RA secretion by cultured macrophages. While soluble CFZ did not affect IL-1RA secretion, phagocytosed CLDIs significantly enhanced the production of IL-1RA in a concentration dependent manner (
Next, it was tested whether soluble CFZ or CLDIs were able to activate pro-inflammatory signaling pathways by monitoring the secretion of TNFα from the cultured cells. Soluble CFZ or CLDIs failed to induce TNFα production on their own (
Inhibition of IκB phosphorylation and NF-κB activation could be achieved by interference with multiple points upstream of the signaling pathway TLR2, TLR4 and TLR9 levels, as well as the adaptor molecule MyD88 were assayed. Increasing concentrations of CLDI treatment led to decreased levels of TLR2 and TLR4 expression following 24 h incubation (
In order to verify whether the accumulation of soluble CFZ or CLDIs altered TLR2 expression in mice, primary alveolar macrophages (AM) from mice fed with control chow or CFZ for 4 wks (which are loaded with soluble CFZ but not CLDIs) or 8 wks (which contain CLDIs) (
Animal Experiments.
Mice (4 week old, male C57Bl6) were purchased from the Jackson Laboratory (Bar Harbor, Me.) and acclimatized for 1 week in a specific-pathogen-free animal facility. Animal care was provided by the University of Michigan's Unit for Laboratory Animal Medicine (ULAM), and the experimental protocol was approved by the Committee on Use and Care of Animals. Clofazimine (CFZ) (C8895; Sigma-Aldrich, St. Louis, Mo.) was dissolved in sesame oil (Roland, China, or Shirakiku, Japan) to achieve a concentration of 3 mg/ml, which was mixed with Powdered Lab Diet 5001 (PMI International, Inc., St. Louis, Mo.) to produce a 0.03% drug to powdered feed (Baik et al., 2013, supra). A corresponding amount of sesame oil was mixed with chow for vehicle treatment. On average, food consumption for a 25 g mouse was 3 g/day, resulting in 10 mg of bioavailable drug/kg per day. For CFZ treatment, the drug diet was carried out for 8 weeks followed by a switch to a control diet for 8 weeks (washout phase).
Measurement of CFZ in Tissues.
CFZ mass was measured as previously reported (Baik et al., 2012, supra; Venkatesan, et al., Arzneim. Forsch. 2007, 57, 472-474). Briefly, at predetermined time points, mice were euthanized using CO2, and blood was removed through cardiac puncture. Organs and tissues were harvested, washed in cold DPBS, and kept at −20° C. until further analysis. The tissues were homogenized with Tissumizer (Tekmar®, Cincinnati, Ohio) and CFZ was extracted with dichloromethane twice followed by solvent evaporation and resolubilization in methanol (MeOH). CFZ mass was spectrophotometrically determined in methanol (λ=490 nm) and the concentration was calculated using a standard curve generated by spiking extracted tissue of the control (vehicle-only treated) mice tissue with known amounts of drug. The tissue-to-fat partition ratios were computed based on mass of drug in the various tissues relative to mass of drug in total body fat (units: dimensionless; calculated as [mg CFZ/g tissue]/[mg CFZ/g fat]). The tissue to plasma partition ratios were computed based on mass of drug in the various tissues relative to mass of drug in plasma volume (units: g−1 tissue, calculated as [mg CFZ/g tissue]/[mg CFZ in blood]). CFZ mass was measured in relation to the measured weight of the tissue or plasma volume.
Isolation of CLDIs from Mouse Spleens.
Spleen tissue homogenate at 8 weeks post drug feeding was sonicated for 30 minutes and clarified by centrifugation (100×g for 1 min). Supernatant was resuspended in 10% sucrose solution in H2O followed by centrifugation (100×g) to remove large cell debris. The drug inclusions in the supernatant were then pelleted by centrifugation (21,000×g for 1 min) and resuspended in 20 ml of 10% sucrose. Clofazimine content was spectrophotometrically (k=490 nm) determined using calibrated CFZ standards (Baik et al., 2013; supra; Baik, J. et al., PLoS One 2012, 7, e47494; Baik, J. et al., Mol. Pharm. 2012, 8, 1742-1749).
Preparation and Transmitted Light Microscopy of Cryopreserved Tissues.
After euthanasia, organs removed from CFZ and vehicle fed mice were cryo-preserved in optimal cutting temperature compound (Tissue-Tek 4583; Sakura). Tissue blocks were sectioned at a thickness 10 using a Leica 3050S cryostat. For transmitted microscopy, cryo-sectioned slices were mounted on glass slides with glycerol, and imaged with Olympus X51 upright microscope equipped with X100 objective, DP-70 color camera, and DP controller 3.1.1267.
Sample Preparation and Transmission Electron Microscopy (TEM).
For TEM, mice were euthanized and they were perfused blood-free by infusing 0.1 M Sorensen's buffer into left ventricle. After flushing for five minutes, five times the total blood volume of fixative containing Karnovsky's solution (3% paraformaldehyde, 2.5% glutaraldehyde) was infused. Afterwards, organs were removed, minced, and kept in the fixative solution at 4° C. until further processing. For staining, the tissues were incubated with osmium tetroxide and dehydrated in alcohol. Dehydrated samples were infiltrated with Epon resin and then polymerized at 60° C. for 24 hours. The polymerized tissue blocks were sectioned with an ultramicrotome and post-stained with uranyl acetate and lead citrate. TEM was performed with a Philips CM-100 instrument equipped with a Hamamatsu ORCA-HR camera system operated by Advanced Microscopy Techniques (Danvers, Mass.).
Synthesis of CFZ Crystals.
CFZ was dissolved in MeOH at 2 mM. Equal volumes of anti-solvents were added to obtain the drug crystals—0.1 M HCl—CFZ-A1, 0.1 M NaOH—CFZ-B, H2O—CFZ-N, 1 M NH4Cl—CFZ-A2. The supernatant was removed, and the crystals were washed and lyophilized in the dark in preparation for further analysis.
Powder X-Ray Diffraction (p-XRD).
Powder XRD of dried samples of isolated CLDIs, 8 week treated (or vehicle) mouse tissue homogenate and synthetic CFZ samples were carried out with Bruker D8 Advance—Cu Kα radiation (λ=1.5406 Å), tube voltage=40 kV, tube current=40 mA. Data were collected at 2θ=4° to 40° at a continuous scan at the rate of 2.5°/min. Diffractograms of the triclinic (DAKXUI01) and monoclinic (DAKXUI) forms of CFZ crystals were imported from Cambridge Structural Database (CSD) and CFZ-TC crystals (C8895; Sigma-Aldrich, St. Louis, Mo.) were used as a positive control for comparison.
Solution NMR.
1D 1H-NMR and 2D HSQC spectra for various CFZ samples and CLDIs were acquired at the University of Michigan's Biochemical NMR Core Laboratory using an 11.74 Tesla (500 MHz) NMR spectrometer with a VNMRS console and a 7510-AS autosampler system operated by host software VNMRJ 3.2, and equipped with a 5 mm Agilent OneNMR probe with Z-axis gradients. DMSO-d6 (100%, 99.9% atom % D) was purchased from Cambridge Isotope Laboratories, Inc. (Tewksbury, Mass.). The samples were prepared by first freeze-drying the samples overnight followed by dissolution in DMSO-d6 at 2 mg/ml. 128 (1H) and 8 (HSQC) scans were acquired for each sample. For accurate identification of the NMR peaks in spectrum, a DMSO-d6-solubilized CFZ spectrum was acquired from a saturated solution of CFZ in DMSO: 1H 64 scans; 13C 9000 scans; HMBC 32 scans; HSQC (gc2hsqcse was the pulse sequence) 8 scans; COSY (DQF-COSY) 8 scans; NOESY 8 scans. All the experiments were run at 25° C. and using the standard parameters from VNMRJ4.0 (Agilent Technologies). All the data were processed with MestreNova 9.0 (MestreLab, Santiago de Compostela, Spain).
Bulk Elemental Composition Analysis (BEA).
CFZ samples were pelleted via centrifugation (10,000×g, 2 min) followed by removal of supernatant and freeze-drying overnight. All samples were sent to Atlantic Microlab, Inc. (Norcross, Ga.) for elemental analysis. Established protocols for the measurement of carbon (C), hydrogen (H), nitrogen (N), chlorine (Cl) and sulfur (S) were used to obtain elemental data. All instrumentation used were calibrated daily with ultra-high purity standards. In brief, samples were accurately weighed using electronic microbalances. C, H, N and S analyses were performed on automatic analyzers based on a modified Pregl and Dumas methodology wherein samples were flash combusted in an oxygen atmosphere at ˜1400° C. Quantitative combustion was achieved by passing the mixture of gases over oxidizing agents comprised of copper oxide, EA1000 (chromium and nickel oxide mixture) and tungstic anhydride, and then over copper, maintained at 650° C., to remove excess oxygen and to reduce the oxides of N. The individual components were separated and eluted as CO2, H2O, N2 and SO2 followed by measurement via a thermal conductivity detector. Cl analysis was performed by Schoniger flask combustion followed by analysis using ion chromatography. The sample was diluted to 25 ml, 50 ml, or 75 ml, filtered and injected into the IC to yield the ppm levels of Cl. All samples were analyzed in duplicate by different technicians.
Secondary Ion Mass Spectroscopy (nanoSIMS).
CFZ-TC and other crystals of CFZ were prepared as mentioned earlier and deposited onto Si wafers and allowed to dry overnight. For CLDI samples, spleens from 8 week CFZ fed mice were isolated and cryo-preserved in optimal cutting temperature compound (Tissue-Tek 4583; Sakura) for histological sectioning as mentioned above. The sections were then scrapped off gently onto a Si wafer. The spatial distribution of various atomic species along the depth of the sample in cross-sectional pattern was mapped with the Cameca NanoSIMS ion micro-probe (CAMECA Instruments, Inc., Madison, Wis., USA). Briefly, with a primary beam of Cs+, focused to a spot-size of 200 nm on the gold-coated surface of the sample, secondary ions of 12C, 16O, 14N, 28Si, 32S, 31P, 35Cl were sputtered from the sample surface and detected simultaneously in multi-collector mode. Elemental composition was obtained by comparing counts with respect to CFZ-TC as a calibration standard for all atomic species.
X-Ray Crystallography.
Diffraction patterns of CFZ crystals were obtained on a Rigaku Saturn 944+ (Cu Kα (λ=1.54178 Å)) at −188° C. Data reduction was performed using CrysAlisPro (Agilent Technologies), and the structure was solved with direct methods using SHELXS27. Structure building, refinement, and electron density map generation were done with SHELXL via the ShelXle GUI (Hübschle et al., J. Appl. Crystallogr. 2011, 44, 1281-1284). Mercury was used to visualize the crystal structure, access CCDC database as well as obtain predicted p-XRD data.
Data Plotting and Statistical Analysis.
All statistical analysis was performed using either ANOVA or Student's t-test. Results were considered significant if p≦0.05. Plots were constructed using Origin 9.0 (OriginLab Corporation, Northampton, Mass., USA) and laid out in figure format using scalable vector graphics format (svg) in either Inkscape (www.inkscape.org) or GIMP (www.gimp.org). NMR plots were constructed in MestreNova 9.0 (MestreLab, Santiago de Compostela, Spain).
Clofazimine (CFZ)—C27H22Cl2N4—is a weakly basic, small molecule chemical agent with a calculated pKa1=2.31 and pKa2=9.29 and is highly lipophilic (clog P˜7). Like other lipophilic weak bases, it has a long retention time within tissues, and exhibits a highly variable pharmacokinetic half-life (t1/2˜70 days in humans, 7 days in mice) (Levy, L. Am. J. Trop. Med. Hyg. 1974, 23, 1097-1109). Upon oral dosing, CFZ exhibits context-dependent pharmacokinetics; while its half-life is in the order of hours to days after an acute dose, upon prolonged oral administration, its half-life is in the order of weeks to months (Levy et al., supra; Banerjee et al., Am. J. Trop. Med. Hyg. 1974, 23, 1110-1115; Nix et al., Tuberculosis 2004, 84, 365-373). In aqueous solution, CFZ predictably exists in neutral, monoprotonated and diprotonated states depending on the solution pH (
To study the intracellular disposition of CFZ upon prolonged oral dosing, CFZ-TC was fed to 4-5 week old C57Bl/6 mice for at least 8 weeks as an oral diet mixed with sesame oil and regular chow (150 mg, 100 ml, 500 g respectively). Following 3-8 weeks of oral administration, CFZ progressively accumulated within macrophages as membrane-bound crystal-like drug inclusions (CLDIs) (Baik et al., 2012, supra; Baik et al., 2013, supra) in the spleen and liver (
To study the sites of CFZ retention, transmitted light microscopy and transmission electron microscopy (TEM) were used to visualize tissue cryo-sections of spleen and liver (
Deep-Etch Freeze-Fracture Electron Microscopy (FFEM) was used to inspect the interface between the internal core of the CLDI and the cytoplasm at high magnification (
Thus, CLDIs reflect an increase in the partition coefficient of CFZ into these tissues, relative to body fat (
The protonation state of the CFZ molecule in the different CFZ crystals and CLDIs was examined via solution NMR studies, conducted in DMSO-d6. 1H NMR of CFZ-A1 and CFZ-A2 revealed a chemical shift of +δa=0.25 ppm and +δb=0.15 ppm for the aliphatic protons (1-7,
Bulk elemental analysis (BEA) was then performed to determine the exact elemental composition of CFZ-A1, CFZ-A2 and CFZ-TC. Using the ratio N/Cl as a direct indicator of the stoichiometry of Cl in relation to the number of protonatable amines, BEA confirmed the presence of an additional Cl in CFZ-A1 and CFZ-A2 relative to CFZ-TC (CFZ-A1 and CFZ-A2 had an N/Cl=1.33 whereas in CFZ-TC, N/Cl=2; Table 1). This confirmed that CFZ-A1 and CFZ-A2 were identical in chemical composition, confirmed to be the monoprotonated hydrochloride salt of CFZ, hereby referenced as CFZ-HCl. Since, CLDIs were derived from a biological source and contained cellular-derived impurities in the form of membranous domains (
To probe the crystal structures of CLDIs in relation to its different crystal forms, powder X-Ray Diffraction (p-XRD) was performed on samples of isolated spleen CLDIs, CFZ-TC, CFZ-A1 and CFZ-A2. The diffraction peak corresponding to 2κ=9.3° (d=9.71 Å) was present exclusively to CFZ-TC (
While the X-ray diffraction signals acquired from single CLDIs were not useful in terms of constructing a 3D molecular model, noting the fact that CLDIs and CFZ-HCl are structurally and chemically similar (
Tables 3-8 show further structural data.
1H NMR Integrals for (a) CFZ—TC, (b) Spleen CLDIs,
7.3
~
7.3
~
9.1
~
9.3
#
9.5
~
10.0
~
11.0
11.5{circumflex over ( )}
11.5{circumflex over ( )}
12.2
#
12.9
13.5
#
14.0{circumflex over ( )}
14.0{circumflex over ( )}
14.7
#
16.1
#
16.2
~
16.8
#
16.8
#
16.9
~
17.2
17.6
~
18.0
#
18.2{circumflex over ( )}
18.2{circumflex over ( )}
18.6{circumflex over ( )}
18.6{circumflex over ( )}
18.9
~
19.2
19.7
19.9
#
19.9
#
20.2
~
20.4
~
21.5
~
21.9{circumflex over ( )}
21.9{circumflex over ( )}
22.2
~
22.7
~
23.2{circumflex over ( )}
23.2{circumflex over ( )}
23.5{circumflex over ( )}
23.5{circumflex over ( )}
24.0{circumflex over ( )}
24.0{circumflex over ( )}
24.5{circumflex over ( )}
24.5{circumflex over ( )}
25.2
~
25.8
~
25.9
26.0
26.1
26.5
#
27.2{circumflex over ( )}
27.2{circumflex over ( )}
28.1
#
28.3{circumflex over ( )}
28.7
~
28.9
#
29.0
~
29.3
#
29.5
30.0
30.2
~
30.5
~
Reagents
Anti-caspase 1 and anti-IL-1β antibodies were purchased from Thermo Pierce (Rockford, Ill.) and Novus Biologicals (Littleton, Colo.), respectively. Anti-actin antibody, LPS (from E. coli 055:B5) and carrageenan was purchased from Sigma (St. Louis, Mo.).
Mice Clofazimine Treatment
Clofazimine (CFZ, Sigma-Aldrich, C8895) was prepared in sesame oil (Shirakiku, Japan, or Roland, China) and Powdered Lab Diet 5001 (PMI International, Inc., St. Louis, Mo.) and orally administrated to Wild-type (WT) C57BL/6 mice or IL-1RA−/− (4-5 week old, Jackson Laboratory, Bar Harbor, Me.) for up to 8 weeks ad libitum as previously described (Baik et al., 2013, supra; Baik et al., 2011, supra). Control mice were fed with the same diet without CFZ. The animal protocol was approved by the University of Michigan's Animal Care and Use Committee in accordance with the National Institutes of Health guidelines (UCUCA #PRO0005111).
Carrageenan Foot Edema Test
Foot inflammation in response to carrageenan injection was conducted in CFZ and control mice as previously described (Otterness I G, et al., Moore P F (1988) Immunochemical Techniques Part L: Chemotaxis and Inflammation (Elsevier) doi:10.1016/0076-6879(88)62086-6). In brief, the volume of each hind paw was measured with a caliper before and after the intraplantar injection of 30 μl 2% carrageenan (60 μg per paw) in PBS or equal volume of PBS in the contralateral paw. Paw swelling was measured at 4 and 48 h after injection, after which the animals were euthanized by exsanguination while deeply anesthetized with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg), and the skin tissues of the plantar region were harvested for cytokine assay (see below).
Acute Lung Injury and Infrared Pulse Oximetry
Since C57BL/6 mice are relatively resistant towards a single dose of intratracheal (IT) LPS instillation (Matute-Bello G et al., 2008 Am J Physiol Lung Cell Mol Physiol 295(3):L379-99), two IT injections of LPS (16 mg/kg; 50 μl) were administered, one on day 0 and the second on day 3. Briefly, 8 wk CFZ-treated and control mice were anesthetized using intraperitoneal injections of xylazine (50 mg/kg) and ketamine (5 mg/kg). Under direct visualization of the vocal cords using an otoscope, either LPS or an equivalent volume of PBS in a 1 mL syringe attached to an oral gavage needle (22 G) was instilled into the lungs via the oral route. The mouse was then placed in a temperature-controlled cage (37° C.) for recovery from anesthesia.
The general health status of each PBS/LPS-instilled mice was monitored by measuring body weight and rectal temperature (Microprobe Thermometer, Physitemp Instruments, Clifton, N.J.), and cardiopulmonary function (arterial oxygen saturation, respiratory rate, heart rate and pulse distention) was monitored using MouseOx with a collar clip sensor (Starr Life Sciences Corp, Oakmont, Pa.) as previously described (Lax S et al., (2014) BMJ open Respir Res 1(1):e000014; Nayak S, et al. (2014) PLoS One 9(6):e98336). In brief, one day before IT instillations (D-1) the hair around the neck of each mouse was removed using Nair (Church & Dwight, Princeton, N.J.) to enable data acquisition using the collar clip sensor. The next day (D 0), immediately prior to the first LPS/PBS dose, and every 24 h afterwards until day 6 (D 6), the body weight, temperature and MouseOx readings were recorded. MouseOx data was acquired by very brief anesthesia of the mouse using 5% isoflurane to facilitate the placement of the collar clip sensor. The mouse was then placed in an enclosed chamber with ambient light and allowed to acclimatize for 5 min at which point the animals had recovered normal activities and physiological readings. Arterial oxygen saturation, breath rate, heart rate and pulse distention measurements were then simultaneously recorded for 6 min (15 readings/sec), and any errors caused by motion during recording were excluded, after which the mean value of each parameter was used for further data analysis.
Terminal Endpoint Assessment
To objectively assess LPS-induced mortality, a multi-parametric scoring system that relied on the daily changes in vital signs associated with inflammatory injury progression and mortality which included arterial oxygen saturation was used (Lax et al., 2014, supra), body weight and temperature (Toth L A (2000) ILAR J 41(2):72-79; Nemzek et al., (2004) Humane endpoints in shock research. Shock 21(1):17-25). First, the percent change from baseline (D 0) in arterial oxygen saturation, body weight, and temperature in each mouse caused by IT instillation of PBS/LPS were measured and calculated daily until day 6 post-instillation. These data were then used in a vector equation to calculate the distance between each LPS-treated mouse and the mean of the PBS-treated mice to assess the terminal endpoint for each LPS-instilled mouse (see Supplemental Information). CFZ-treated and control mice were calculated separately and LPS-instilled mice that scored a total of 18 or higher were determined as terminal, as these mice also displayed severe signs of sickness evidenced by impaired mobility, lack of grooming, hunched posture, and muscle weakness could be felt while handling the mice. These mice were immediately euthanized with ketamine/xylazine and the bronchoalveolar lavage (BAL) and lungs were harvested for cellular and biochemical analysis (see below). Remaining mice that did not reach terminal endpoint were all euthanized on day 6 post-PBS/LPS instillation.
Mouse Bronchoalveolar Lavage (BAL) Harvest and Isolation of Alveolar Macrophages, Immunohistochemistry and Imaging, SDS-PAGE and Western Blot, Cytokine Measurements
PBS/LPS-instilled CFZ- or control mice were euthanized by exsanguination while deeply anesthetized with an intraperitoneal injection of ketamine/xylazine. The trachea was surgically exposed and cannulated with an 18 G needle and the lungs were lavaged by instilling 1 ml DPBS (Life Technologies) containing 0.5 mM EDTA (Sigma). The retrieved BAL was then centrifuged (10 min at 400×g, 4° C.), and the supernatant was frozen (−80° C.), while the cell pellets were resuspended in 1 ml RPMI 1640 media (Life Technologies). To count total cells, the cells were stained with Trypan blue and counted using a hemocytometer. To distinguish cell types, an aliquot of the cells were dried on glass slides and stained with Diff Stain kit (IMEB Inc, San Marcos, Calif.) according to the manufacturer's instructions. To isolate alveolar macrophages, the cells were placed in 12-well culture plate (Corning, Tewksbury, Mass.) and washed with media after 45 min, enabling the isolation of alveolar macrophages by adherence.
Data Processing and Statistics
All data are expressed as mean±standard deviation (S.D.). For multiple comparisons, statistical analysis was performed with one-way analysis of variance (ANOVA) and Bonferroni's post-hoc comparisons. For two group comparisons an unpaired Student's t-test was used. All statistical analyses employed the Sigmaplot version 13 software and p≦0.05 was considered statistically significant.
Clofazimine Crystallization and Bioaccumulation in the Liver Occurs after 2 Weeks
In the first two weeks of CFZ treatment, CFZ diffusely distributed throughout the liver (
Clofazimine Bioaccumulation and Crystal Formation in the Liver Inhibits Inflammasome Activity and IL-1β Maturation while Enhancing IL-1RA Expression
The formation of insoluble drug precipitates or crystals inside cells is generally considered an adverse drug reaction, since intracellular crystals such as those formed by cholesterol or uric acid have been implicated in the activation of the NLRP3-caspase 1 inflammasome, which plays a pivotal role in the pathogenesis of chronic inflammatory disorders such as atherosclerosis (Duewell P, et al. (2010) Nature 464(7293):1357-61), non-alcoholic steatohepatitis (Ioannou G N et al., (2013) J Lipid Res 54(5):1326-34) and gout (Martinon F et al., (2006) Nature 440(7081):237-41). Uptake of other nano- and micro-particles has also been reported to cause inflammasome activation in macrophages (Simard J-C et al., (2015) J Biol Chem 290(9):5926-39; Demento S L, et al. (2009) Vaccine 27(23):3013-21; Peeters P M, et al. (2014) Part Fibre Toxicol 11:58). Yet, in spite of such concerns, there have not been any studies on the impact of CFZ crystal bioaccumulation on macrophage inflammasome proteins activity. Therefore, to understand whether soluble CFZ or the bioaccumulation of CFZ crystals alters the inflammasome, associated proteins in organs (liver, spleen and lung) known to form CFZ crystals and the kidneys were assessed, which do not, after 2 and 8 weeks of either CFZ or control treatment.
Two weeks of CFZ resulted in only moderate cleavage of hepatic caspase 1 and IL-1β (
Following previous observations that CFZ elevated the endogenous anti-inflammatory signaling molecule interleukin 1-receptor antagonist (IL-1RA) (Baik et al., 2013, supra), it was observed that CLDIs in liver, spleen and lung after 8 weeks of oral administration caused major upregulation of IL-1RA expression in these organs, especially in the liver, whereas IL-1RA levels in the kidney were unchanged (
Since 8 wk CFZ-treated mice displayed high serum IL-1RA levels, the acute inflammatory response of CFZ-treated mice was tested by employing a well-established footpad injury model using an intraplantar injection of carrageenan. CFZ-treated and carrageenan-injected paws displayed strikingly reduced swelling 48 h post-injection compared to the paws of untreated mice (
To test whether the reduced inflammatory response observed in 8 wk CFZ treated mice was associated with CFZ crystal formation, carrageenan-induced footpad inflammation experiments were performed using mice that received 2 weeks of CFZ treatment (Baik et al., 2013, supra). At 48 h post-carageenan injection, only a modest reduction in paw swelling was observed compared with untreated mice (
IL-1RA is known to be an early-acting acute-phase anti-inflammatory cytokine (Gabay et al., 1997, supra), and circulating IL-1RA has been reported to dampen a broad spectrum of inflammatory conditions (Dinarello C A, van der Meer J W M (2013) Semin Immunol 25(6):469-84). Its role is likely to inhibit IL-1β activity from spreading beyond a certain inflamed area while dampening the intensity of the inflammatory response (Arend W P (2002) Cytokine Growth Factor Rev 13(4-5):323-340). To see how serum IL-1RA levels changed following carrageenan injection into the paw, IL-1RA was detected in the serum of naïve (0 h), carrageenan-injected mice at 48 h and/or 4 h post-injection. Prior to the onset of CLDI formation, IL-1RA levels in the serum of control and 2 week CFZ treated mice were similar to those of uninjured mice. Comparing 2 week CFZ treated mice with untreated mice showed that IL-1RA increased to similar levels at 48 h post carrageenan injury (
In order to further explore the possibility that elevated IL-1RA expression was mediating the anti-inflammatory activity of CFZ following CLDI formation, CFZ was given to age-matched IL-1RA KO and WT mice for 6 weeks, and tested using the footpad injury model. These experiments were done at 6 weeks and not at 8 weeks, because the IL-1RA KO mice weighed less than WT and their livers were smaller (
Given the massive bioaccumulation of CFZ observed in vital organs such as the liver, spleen and lungs, it was determined whether the presence of CLDIs in these organs may sensitize the mice to a lethal pro-inflammatory injury. First, daily arterial oxygen saturation was measured as an indicator of lung function, as well as body weight, temperature, heart rate, breathing rate and pulse distention (measure of arterial blood flow, pulse pressure) to measure overall health. Baseline measurements of 8 wk CFZ-treated and control diet-treated mice show that 8 wk CFZ-treated mice weighed 6.7% less, and displayed lower breathing rate (10.7%), heart rate (9.3%) and pulse distention (16.9%) (Table 9). Arterial oxygen saturation was 1.5% higher in CFZ-treated than their control diet-treated counterparts (Table 9), indicating that lung function was normal or even enhanced by CFZ bioaccumulation. Body temperature was unaffected by CFZ treatment. Also, CFZ-treated mice did not display any behavioral signs of sickness as mobility, grooming, and posture were normal, and no signs of muscle weakness could be felt while handling the mice. These results indicate that although prolonged CFZ treatment and bioaccumulation caused certain changes in physiological parameters, these changes were not outside the ranges of normal physiological readings and seemed that CFZ bioaccumulation did not have any adverse effects on mouse overall health.
Next, the effect of a lethal dose of LPS on lung function and health of control diet-treated and CFZ-treated mice was evaluated. CFZ-treated mice were highly resistant to LPS-induced acute lung injury with a 92% survival rate compared to control diet-treated mice (42% survival rate) (
All publications and patents mentioned in the present application are herein incorporated by reference. Various modification and variation of the described methods and compositions of the disclosure will be apparent to those skilled in the art without departing from the scope and spirit of the disclosure. Although the disclosure has been described in connection with specific preferred embodiments, it should be understood that the disclosure as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the disclosure that are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.
The present application claims priority to U.S. Provisional Patent Application Ser. No. 62/109,906 filed Jan. 30, 2015, and U.S. Provisional Patent Application Ser. No. 62/111,921 filed Feb. 4, 2015, each of which is hereby incorporated by reference in its entirety.
This invention was supported in part by Grant Nos. R01GM078200 and R01AR060350 awarded by the National Institutes of Health. The government may have certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62109906 | Jan 2015 | US | |
| 62111921 | Feb 2015 | US |
