WO2015153864A2

WO2015153864A2 - Methods for treating inflammatory conditions

Info

Publication number: WO2015153864A2
Application number: PCT/US2015/024047
Authority: WO
Inventors: Patricia T. HOPKINS
Original assignee: Hopkins Patricia T
Priority date: 2014-04-02
Filing date: 2015-04-02
Publication date: 2015-10-08
Also published as: WO2015153864A3

Abstract

The invention features methods of treating subjects for inflammatory conditions, such as mild traumatic brain injury (mTBI), osteoarthritis (OA), and fibromyalgia. The method may involve determining if the subject has such an inflammatory condition and administering an effective amount of a tetracycline compound (e.g., doxycycline) to the subject (e.g., a human). The invention also features methods for detecting an inflammatory condition in a subject by determining a level of an inflammatory biomarker, such as TGF-βΙ, in a biological sample (e.g., blood) obtained from the subject. Such methods can also be used as a companion diagnostic in combination with tetracycline treatment to assess treatment efficacy. The invention further features kits for detecting and treating such inflammatory conditions.

Description

METHODS FOR TREATING INFLAMMATORY CONDITIONS

Field of the Invention

The present invention relates to treating inflammatory conditions in subjects, such as mild traumatic brain injury (mTBI) and osteoarthritis.

Background

Inflammation is a component of the innate immune response and is associated with a number of disorders. Inflammatory conditions include mild traumatic brain injury (mTBI), also known as concussion, and osteoarthritis, both of which are primarily treated through physical therapy and palliative care.

mTBI is a complex pathophysiological process affecting the brain, which occurs due to unwanted exposure to traumatic biomechanical forces (Patterson and Holahan, Front. Cell Neurosci., 6(58): 1 -10, 2012). It results in transient impairment of neurological function that often improves with time.

Importantly, brain imaging does not reveal gross structural changes after mTBI, thus making the entire diagnosis clinical in nature. mTBI commonly occurs as a result of sports injury and is increasingly being identified in the military and battlefield combat setting. The symptoms of mTBI are myriad and nonspecific and include headache, nausea, vomiting, fatigue, alteration of sleep cycles, and in more severe cases, drowsiness, alteration of consciousness and neurocognitive changes (Bryan, Sleep, 36(6): 941 - 946, 2013, Theeler et al., Headache, 53(6): 881 -900, 2013; Maroon et al., Phys. Sportsmed., 40(4): 73- 87, 2012). mTBI often occurs multiple times in the same subject. Repetitive brain injuries can seriously and negatively impact patients in the long term, leading to changes in personality, sleep problems, and cognitive impairments, and can increase the risk for suicide, post-traumatic stress disorder, depression, and anxiety. In some people, repetitive mTBI can lead to chronic traumatic encephalopathy (CTE), a neurodegenerative disorder.

It has been hypothesized that mTBI may result from biomechanical rotational forces that result in rapid changes in acceleration and decelerations of the brain often leading to functional impairments in the absence of a visual damage to brain architecture. The stretching and shearing of axonal and cell membranes lead to diffuse neuronal damage mainly due to disruption in the axonal segment, further resulting in ionic disequilibrium and metabolic stress. The time course of injury is also not well known but it is generally presumed that much of the damage suffered from mTBI occurs due to the delayed progression of secondary biochemical events which ultimately lead to neuronal dysfunction. Recent evidence suggests that immunoexcitotoxicity is a primary basis for disease pathophysiology (Patterson and Holahan, Front. Cell Neurosci., 6(58): 1 -10, 2012).

The use of a biological correlate at or near the site of injury, such as the Cerebrospinal Fluid

(CSF), is potentially advantageous to examine neural injury after mTBI. However, the acquisition of CSF fluid is a relatively invasive procedure in comparison to obtaining a blood sample from a patient population. No clinically accepted TBI peripheral blood biomarkers currently exist (Bettermann and Slocomb, Biomarkers for Traumatic Brain Injury, Chapter 1 , "Clinical relevance of biomarkers for traumatic brain injury," 1 -18, 2012). In specific populations such as military personnel, the relationship between combat-related mTBI and residual mTBI symptoms, post-traumatic stress disorder (PTSD) symptoms, and neurocognitive deficits remains unclear (Brenner, Dialogues Clin. Neurosci., 13: 31 1 - 323, 201 1 ; Miller, Science, 333: 514-517, 201 1 ).

The economic effect of mTBI is substantial, accounting for approximately 44% of the US$60 billion annual cost of TBI in the USA. However, the relationship between combat-related mTBI and residual mTBI symptoms resulting from blast and/or blunt injuries, post-traumatic stress disorder (PTSD) symptoms, and neurocognitive deficits remains unclear. mTBI resulting from mild traumatic brain injury is increasingly being reported in returning military personnel and is a huge cost burden (Bryan and

Clemans, J.A.M.A. Psychiatry, 70(7): 686-691 , 2013; Nowinski, Cerebrum, 2, 2013; Peskind et al., J. Clin. Psychiatry, 74(2): 180-188, 2013; Lange et al., J. Neurotrauma, 30(4): 237-246, 2013; Kennedy et al., Arch. Clin. Neuropsychoi, 27(8): 817-827, 2012; Kontos et al., J. Neurotrauma, 30(8): 680-686, 2013;

Rosenfeld et al., Lancet Neurol., 12(9): 882-893, 2013). Reliable biomarkers of progression of symptoms after mTBI are not available (Di Battista et al., Front. Neurol., 4: 44, 2013; Dash et al., Neurotherapeutics, 7: 100-1 14, 2010).

Osteoarthritis (OA) is the most common form of arthritis, affecting nearly 27 million people in the United States, and is a chronic degenerative disorder affecting joint tissue in, e.g., the hands, feet, hips, knees, or spine. Fibrosis of the surrounding muscle architecture has been implicated in the development of advanced osteoarthritis.

Doxycycline is a semisynthetic, chemically modified tetracycline compound that is rapidly absorbed when taken orally and topically, and which exerts biological effects that are independent of antimicrobial activity (Greenwald, Ann. N. Y. Acad. Sci., 732: 181 -198, 1994).

Although a number of investigated therapies against inflammatory conditions, such as mTBI or OA, have demonstrated palliative properties, these therapies may not produce consistently beneficial outcomes in subjects. Therefore, there remains a need for methods to treat subjects with such inflammatory conditions as well as methods to accurately diagnose the presence of such inflammatory conditions in subjects.

Summary of the Invention

The invention features methods and kits for treating subjects with inflammatory conditions, as well as methods for detecting inflammatory conditions. Such inflammatory conditions may include, for example, mild traumatic brain injury (mTBI), also known as concussion, osteoarthritis (OA), fibromyalgia, diabetic adhesive capsulitis, hypermobility syndrome, exercise intolerance, arthropathy, inflammatory bowel disease, constipation, diabetes-associated inflammation, adhesion (e.g., post-operative adhesion, post-operative adhesion after joint replacement surgery, or abdominal adhesion), Ehlers-Danlos

Syndrome, sarcopenia, sarcoidosis, and liver fibrosis. In various embodiments, the adhesion is the result of injury, surgery (e.g., joint replacement surgery), or inflammation. In some embodiments, the adhesion occurs in a joint, tendon, ligament, eye, abdomen, pericardium, or pelvis. In certain embodiments, the joint is a shoulder joint, knee joint, hip joint, ankle joint, elbow joint, or wrist joint. In alternate

embodiments, the adhesion occurs in the abdomen (e.g., an abdominal adhesion) and is further associated with intestinal blockage, bloating, constipation, nausea, vomiting, abdominal pain, or cramps. In further embodiments, the adhesion occurs in the eye (e.g., an eye adhesion) and is further associated with glaucoma. In a first aspect, the invention features a method of treating a subject having an elevated TGF-β level (e.g., associated with an inflammatory condition) by administering an effective amount of a tetracycline to the subject, e.g., in which the inflammatory condition results from a condition other than a bacterial infection.

In some embodiments of the first aspect, the subject has an elevated TGF-β level (e.g., TGF-βΙ ,

TGF^2, or TGF^3). In specific embodiments, the TGF-β is TGF-βΙ . In certain embodiments, the level of TGF-β is determined in a biological sample obtained from the subject (e.g., blood, peripheral blood, a blood component (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid (CSF), synovial fluid, tissue (e.g., placental or dermal), pancreatic fluid, chorionic villus sample, and cells). In a specific embodiment, the biological sample is or includes blood.

In some embodiments of the method, the presence of an inflammatory condition can be determined using methods known in the art for diagnosing an inflammatory condition and/or by assaying the level of at least one inflammatory biomarker (e.g., those listed in Table 1 below, such as, for example, MMP1 , MMP3, TGF-βΙ , TGF^2, or TGF^3; preferably TGF-βΙ ) in a biological sample from the subject (e.g., blood, peripheral blood, a blood component (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid (CSF), synovial fluid, tissue (e.g., placental or dermal), pancreatic fluid, chorionic villus sample, and cells; preferably blood, cerebrospinal fluid, or synovial fluid).

Table 1. Inflammatory biomarkers (e.g., cytokines) for which elevated levels, relative to that of a normal subject, indicate the presence of an inflammatory condition.

The inflammatory condition can result from a condition other than bacterial infection. In certain embodiments, the subject has a sterile inflammation.

In some embodiments of the method, the inflammatory condition is selected from the group consisting of mTBI, OA, fibromyalgia, diabetic adhesive capsulitis, hypermobility syndrome, exercise intolerance, arthropathy, inflammatory bowel disease, constipation, diabetes-associated inflammation, adhesion (e.g., post-operative adhesion, post-operative adhesion after joint replacement surgery, or abdominal adhesion), Ehlers-Danlos Syndrome, sarcopenia, sarcoidosis, and liver fibrosis. For example, the inflammatory condition may be mTBI, OA, fibromyalgia, constipation, or adhesion. In various embodiments, the adhesion is the result of injury, surgery (e.g., joint replacement surgery), or inflammation. In particular embodiments, the adhesion occurs in a joint, tendon, ligament, eye, abdomen, pericardium, or pelvis. In specific embodiments, the joint is a shoulder joint, knee joint, hip joint, ankle joint, elbow joint, or wrist joint. In alternate embodiments, the adhesion occurs in the abdomen (e.g., an abdominal adhesion) and is further associated with intestinal blockage, bloating, constipation, nausea, vomiting, abdominal pain, or cramps. In further embodiments, the adhesion occurs in the eye (e.g., an eye adhesion) and is further associated with glaucoma.

In a second aspect, the invention features a method of treating a subject having an inflammatory condition selected from the group consisting of: mild traumatic brain injury (mTBI), osteoarthritis (OA), fibromyalgia, diabetic adhesive capsulitis, hypermobility syndrome, exercise intolerance, arthropathy, inflammatory bowel disease, constipation, diabetes-associated inflammation, adhesion (e.g., postoperative adhesion, post-operative adhesion after joint replacement surgery, or abdominal adhesion), Ehlers-Danlos Syndrome, sarcopenia, sarcoidosis, and liver fibrosis (e.g., mTBI, OA, fibromyalgia, constipation, or adhesion) by administering to the subject an effective amount of a tetracycline (e.g., doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK-SMA2; preferably doxycycline or sarecycline). In an embodiment, the method excludes the treatment of a subject having osteoarthritis. In another embodiment, the method excludes treatment with minocycline or minocycline derivatives. In still other embodiments, the method excludes the treatment of a subject having osteoarthritis with minocycline or minocycline derivatives.

In an embodiment of the second aspect, the method further includes, prior to the administering step, determining if the subject has the inflammatory condition (e.g., by using methods known in the art for diagnosing an inflammatory condition and/or by assaying the level of at least one inflammatory biomarker in a biological sample from the subject according to the methods of the invention described herein). For example, the inflammatory condition may be mTBI, OA, fibromyalgia, constipation, or adhesion (e.g., post-operative adhesion, post-operative adhesion after joint replacement surgery, or abdominal adhesion). In various embodiments, the adhesion is the result of injury, surgery (e.g., joint replacement surgery), or inflammation. In particular embodiments, the adhesion occurs in a joint, tendon, ligament, eye, abdomen, pericardium, or pelvis. In specific embodiments, the joint is a shoulder joint, knee joint, hip joint, ankle joint, elbow joint, or wrist joint. In alternate embodiments, the adhesion occurs in the abdomen (e.g., an abdominal adhesion) and is further associated with intestinal blockage, bloating, constipation, nausea, vomiting, abdominal pain, or cramps. In further embodiments, the adhesion occurs in the eye (e.g., an eye adhesion) and is further associated with glaucoma.

In some embodiments of any of the above methods, the tetracycline (e.g., doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK-SMA2, another tetracycline described herein, or a derivative thereof; preferably doxycycline or sarecycline) is administered orally, transdermally, topically, intravenously, or by injection. In an embodiment, the tetracycline is administered orally. Preferably, the tetracycline is doxycycline that is administered orally. In some embodiments, the tetracycline is doxycycline that is administered transdermally. In other embodiments, the tetracycline is doxycycline that is administered topically.

In certain embodiments of the method, a single dose of the tetracycline (e.g., doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK-SMA2, another tetracycline described herein, or a derivative thereof; preferably doxycycline or sarecycline ) is administered in an amount between 1 mg and 1000 mg. In preferred embodiments, the dose of the tetracycline

administered is an amount between 75 mg and 300 mg, e.g., about 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 1 10 mg, 1 15 mg, 120 mg, 125 mg, 130 mg, 135 mg, 140 mg, 145 mg, 150 mg, 155 mg, 160 mg, 165 mg, 170 mg, 175 mg, 180 mg, 185 mg, 190 mg, 195 mg, 200 mg, 205 mg, 210 mg, 215 mg, 220 mg, 225 mg, 230 mg, 235 mg, 240 mg, 245 mg, 250 mg, 255 mg, 260 mg, 265 mg, 270 mg, 275 mg, 280 mg, 285 mg, 290 mg, 295 mg, or 300 mg. In some embodiments, the tetracycline is administered at a submicrobial dose.

In certain embodiments of the method, the tetracycline (e.g., doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK-SMA2, another tetracycline described herein, or a derivative thereof; preferably doxycycline or sarecycline; preferably doxycycline) is administered one or more times (e.g., at least once) in intervals of 1 -48 hours, e.g., about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 1 1 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 30 hours, 36 hours, 40 hours, or 48 hours. In preferred embodiments, the tetracycline is administered at least once every 12 or 24 hours. In some embodiments, the tetracycline is administered at least once daily for 1 day to 1 year, e.g., about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 1 1 months, or 1 year. In preferred embodiments, the tetracycline is administered at least once daily for 1 week to 3 months, e.g., about 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, or 3 months. In particular embodiments, the tetracycline is administered at least once daily for about 1 week, 1 month (30 days), or 3 months. Preferably the tetracycline is administered for about 1 week, 1 month (30 days), or 3 months at a dose in the amount of about 100 mg.

The tetracycline may be selected from the group consisting of doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK-SMA2, tetracycline, chlortetracycline, oxytetracycline, demeclocycline, lymecycline, meclocycline, methacycline, rolitetracycline, clomocycline, metacycline, pipacycline, incyclinide, or a derivative thereof. In preferred embodiments, the tetracycline is doxycycline, minocycline, sarecycline, or omadacycline. In specific embodiments, the tetracycline is doxycycline or sarecycline.

In an exemplary embodiment, doxycycline is administered in an amount of 100 mg at least once daily for at least 30 days.

In certain embodiments of the method, the method further involves administering a second therapeutic agent. In particular embodiments, the second therapeutic agent is selected from the group consisting of a non-steroidal anti-inflammatory drug (NSAID), a COX-2 inhibitor, capsaicin, and gabapentin.

In a third aspect, the invention features a method of predicting whether a subject with an inflammatory condition would be responsive to treatment with a tetracycline. The method involves determining a level of at least one inflammatory biomarker, for example, an inflammatory cytokine selected from one or more of the inflammatory cytokines in Table 1 (e.g., TGF-βΙ , TGF^2, or TGF^3), in a biological sample taken from the subject. A determination that the level of the inflammatory biomarker in the biological sample is elevated, relative to a level of the inflammatory biomarker in a normal subject, indicates that the subject would be responsive to treatment with the tetracycline.

In a fourth aspect, the invention features a method for treating an inflammatory condition in a subject by determining a level of a TGF-β (e.g., TGF-βΙ , TGF^2, or TGF^3) in a biological sample taken from the subject. A determination that the level of the TGF-β in the biological sample is elevated, relative to the level in a normal subject, indicates the presence of the inflammatory condition. If the presence of the inflammatory condition is detected in the subject, then an effective amount of a tetracycline (e.g., doxycycline, minocycline, sarecycline, or omadacycline; preferably doxycycline or sarecycline) is administered to the subject.

In some embodiments of the fourth aspect, the TGF-β is TGF-βΙ , TGF^2, or TGF^3. In a specific embodiment, the TGF-β is TGF-βΙ and the level in the normal subject is about 20 ng/ml.

In certain embodiments of the method, the tetracycline (e.g., doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK-SMA2, another tetracycline described herein, or a derivative thereof; preferably doxycycline or sarecycline) is administered orally, transdermally, topically, intravenously, or by injection. In a preferred embodiment, the tetracycline is administered orally.

In an embodiment of the third aspect, a single dose of the tetracycline (e.g., doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK-SMA2, another tetracycline described herein, or a derivative thereof; preferably doxycycline or sarecycline) is administered in an amount between 1 mg and 1000 mg. In preferred embodiments, the dose of the tetracycline

administered is an amount between 75 mg and 300 mg, e.g., about 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 1 10 mg, 1 15 mg, 120 mg, 125 mg, 130 mg, 135 mg, 140 mg, 145 mg, 150 mg, 155 mg, 160 mg, 165 mg, 170 mg, 175 mg, 180 mg, 185 mg, 190 mg, 195 mg, 200 mg, 205 mg, 210 mg, 215 mg, 220 mg, 225 mg, 230 mg, 235 mg, 240 mg, 245 mg, 250 mg, 255 mg, 260 mg, 265 mg, 270 mg, 275 mg, 280 mg, 285 mg, 290 mg, 295 mg, or 300 mg. For example, the tetracycline is administered at a submicrobial dose.

In several embodiments of the method, the tetracycline (e.g., doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK-SMA2, another tetracycline described herein, or a derivative thereof; preferably doxycycline or sarecycline) is administered one or more times in intervals of 1 -48 hours, e.g., about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 1 1 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 30 hours, 36 hours, 40 hours, or 48 hours. In preferred embodiments, the tetracycline is administered at least once every 12 or 24 hours. In some embodiments, the tetracycline is administered at least once daily for 1 day to 1 year, e.g., about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 1 1 months, or 1 year. In certain embodiments, the tetracycline is administered at least once daily for 1 week to 3 months, e.g., about 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, or 3 months. In preferred embodiments, the tetracycline is administered at least once daily for about 1 week, 1 month (30 days), or 3 months.

Preferably the tetracycline is administered for about 1 week, 1 month (30 days), or 3 months at a dose in the amount of about 100 mg.

The tetracycline may be selected from the group consisting of doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK-SMA2, tetracycline, chlortetracycline, oxytetracycline, demeclocycline, lymecycline, meclocycline, methacycline, rolitetracycline, clomocycline, metacycline, pipacycline, incyclinide, and a derivative thereof. In preferred embodiments, the tetracycline is doxycycline, minocycline, sarecycline, or omadacycline. In specific embodiments, the tetracycline is doxycycline or sarecycline. In an exemplary embodiment, doxycycline is administered in an amount of 100 mg at least once daily for at least 30 days.

In an embodiment, the method further involves administering a second therapeutic agent (e.g., an agent selected from the group consisting of a non-steroidal anti-inflammatory drug (NSAID), a COX-2 inhibitor, capsaicin, and gabapentin).

In some embodiments, the tetracycline has not previously been administered to the subject to treat the inflammatory condition. In other embodiments, the tetracycline has been previously

administered to the subject to treat the inflammatory condition.

In a preferred embodiment, the biological sample is or comprises blood.

In some embodiments of the fourth aspect, the determining step further includes determining a level of at least one additional inflammatory biomarker (e.g., an inflammatory biomarker listed in Table 1 ) in a biological sample obtained from the subject; in which an elevated level of the at least one additional inflammatory biomarker in the biological sample, relative to a level in a normal subject, indicates the presence of the inflammatory condition.

In an embodiment, at least one of the additional inflammatory biomarkers is selected from the group consisting of MMP1 , MMP3, MMP9, IL1 A, IL1 B, TNFa, IL-6, RANKL, MCP-1 , interferon-gamma, IL- 8, CX3CL1 , CXCL1 , CXCL10, SDF-1 , IL-2, IL-17, IL-18, and IL-23. In other embodiments, a level of the inflammatory biomarker greater than that considered to be a normal level for that biomarker (see Table 1 ) indicates the presence of an inflammatory condition. In other embodiments, a level of the inflammatory biomarker greater than that corresponding to mild, intermediate, or high inflammation for that biomarker (see Table 1 ) indicates the presence of an inflammatory condition.

In a particular embodiment, the at least one additional inflammatory biomarkers is MMP1 and the level in the normal subject is <30 ng/ml. In another embodiment, the at least one of the additional inflammatory biomarkers is MMP3 and the level in the normal subject is <40 ng/ml. In another embodiment, the at least one of the additional inflammatory biomarkers is IL1 B and the level in the normal subject is <80 pg/ml. In another embodiment, the at least one of the additional inflammatory biomarkers is TNFa and the level in the normal subject is <10 pg/ml. In another embodiment, the at least one of the additional inflammatory biomarkers is interferon-gamma and the level in the normal subject is <10 pg/ml. In another embodiment, the at least one of the additional inflammatory biomarkers is IL1 A and the level in the normal subject is <5 pg/ml. In another embodiment, the at least one of the additional inflammatory biomarkers is IL-6 and the level in the normal subject is <10 pg/ml. In another embodiment, the at least one of the additional inflammatory biomarkers is IL-8 and the level in the normal subject is <10 pg/ml. In another embodiment, the at least one of the additional inflammatory biomarkers is CX3CL1 and the level in the normal subject is <1 ng/ml. In each of the embodiments disclosed above, a level of the noted inflammatory biomarker in a sample from the subject that is equal to or greater than the normal level for the biomarker indicates the presence of an inflammatory condition.

In some embodiments of the fourth aspect, the inflammatory condition is selected from the group consisting of mTBI, OA, fibromyalgia, diabetic adhesive capsulitis, hypermobility syndrome, exercise intolerance, arthropathy, inflammatory bowel disease, adhesion (e.g., post-operative adhesion, post- operative adhesion after joint replacement surgery, or abdominal adhesion), constipation, diabetes- associated inflammation, Ehlers-Danlos Syndrome, sarcopenia, sarcoidosis, and liver fibrosis. In preferred embodiments, the inflammatory condition is mTBI, osteoarthritis, fibromyalgia, constipation, or adhesion (e.g., post-operative adhesion, post-operative adhesion after joint replacement surgery, or abdominal adhesion). In various embodiments, the adhesion is the result of injury, surgery (e.g., joint replacement surgery), or inflammation. In some embodiments, the adhesion occurs in a joint, tendon, ligament, eye, abdomen, pericardium, or pelvis. In certain embodiments, the joint is a shoulder joint, knee joint, hip joint, ankle joint, elbow joint, or wrist joint. In alternate embodiments, the adhesion occurs in the abdomen (e.g., an abdominal adhesion) and is further associated with intestinal blockage, bloating, constipation, nausea, vomiting, abdominal pain, or cramps. In further embodiments, the adhesion occurs in the eye (e.g., an eye adhesion) and is further associated with glaucoma.

In a fifth aspect, the invention features a method for treating an inflammatory condition selected from the group consisting of mild traumatic brain injury (mTBI), fibromyalgia, diabetic adhesive capsulitis, hypermobility syndrome, exercise intolerance, arthropathy, inflammatory bowel disease, constipation, diabetes-associated inflammation, adhesion (e.g., post-operative adhesion, post-operative adhesion after joint replacement surgery, or abdominal adhesion), Ehlers-Danlos Syndrome, sarcopenia, sarcoidosis, and liver fibrosis, in a subject. The method includes:

(i) determining a level of an inflammatory biomarker (e.g., an inflammatory biomarker listed in Table 1 ), in which an elevated level of the inflammatory biomarker in the biological sample, relative to a level in a normal subject, indicates the presence of the inflammatory condition; and

(ii) administering an effective amount of a tetracycline to the subject if the presence of the inflammatory condition is detected in the subject.

In a sixth aspect, the invention features a method of treating a subject, in which the following steps are performed:

(i) detecting the presence of an inflammatory condition in the subject;

(ii) determining a level of at least one inflammatory biomarker (e.g., a cytokine)

selected from one or more of the inflammatory biomarkers shown in Table 1 (e.g.,

TGF-βΙ , TGF^2, TGF^3, MMP1 , or MMP3; preferably TGF-βΙ ) in a biological sample obtained from the subject, in which an elevated level of the inflammatory biomarker in the biological sample, relative to a level of the inflammatory biomarker in a normal subject, indicates that the subject would be responsive to treatment with a tetracycline; and

(iii) if the level of the inflammatory biomarker is elevated, relative to the level of the inflammatory biomarker in the normal subject (e.g., if the TGF-β is TGF-βΙ , the level in a normal subject can be about 20 ng/ml), administering an effective amount of the tetracycline to the subject based on the level of the inflammatory biomarker in the biological sample.

In a seventh aspect, the invention features a method for treating an inflammatory condition in a subject by:

(i) determining a level of at least one inflammatory biomarker (e.g., a cytokine), such as those shown in Table 1 (e.g., TGF-βΙ , TGF^2, TGF^3, MMP1 , or MMP3; preferably TGF-βΙ ), in a biological sample obtained from the subject, in which an elevated level of the at least one inflammatory biomarker in the biological sample, relative to a level in a normal subject (e.g., if the TGF-β is TGF-βΙ , the level in a normal subject can be about 20 ng/ml), indicates the presence of the inflammatory condition; and

(ii) administering an effective amount of a tetracycline (e.g., doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK-SMA2, another tetracycline described herein, or a derivative thereof; preferably doxycycline or sarecycline) to the subject if the presence of the inflammatory condition is detected in the subject. In some embodiments of any of the above aspects, the inflammatory biomarker is MMP1 and the level in the normal subject is <30 ng/ml. In other embodiments, the inflammatory biomarker is MMP3 and the level in the normal subject is <40 ng/ml. In other embodiments, the inflammatory biomarker is IL1 B and the level in the normal subject is <80 pg/ml. In other embodiments, the inflammatory biomarker is TNFa and the level in the normal subject is <10 pg/ml. In other embodiments, the inflammatory biomarker is interferon-gamma and the level in the normal subject is <10 pg/ml. In other embodiments, the inflammatory biomarker is IL1 A and the level in the normal subject is <5 pg/ml. In other embodiments, the inflammatory biomarker is IL-6 and the level in the normal subject is <10 pg/ml. In other

embodiments, the inflammatory biomarker is IL-8 and the level in the normal subject is <10 pg/ml. In other embodiments, the inflammatory biomarker is CX3CL1 and the level in the normal subject is <1 ng/ml. In each of the embodiments disclosed above, a level of the noted inflammatory biomarker in a sample from the subject that is equal to or greater than the normal level for the biomarker indicates the presence of an inflammatory condition.

In some embodiments of any of the above aspects, the inflammatory condition is selected from the group consisting of mTBI, OA, fibromyalgia, diabetic adhesive capsulitis, hypermobility syndrome, exercise intolerance, arthropathy, inflammatory bowel disease, adhesion (e.g., post-operative adhesion, post-operative adhesion after joint replacement surgery, or abdominal adhesion), constipation, diabetes- associated inflammation, Ehlers-Danlos Syndrome, sarcopenia, sarcoidosis, and liver fibrosis. In preferred embodiments, the inflammatory condition is mTBI, OA, fibromyalgia, constipation, or adhesion (e.g., post-operative adhesion, post-operative adhesion after joint replacement surgery, or abdominal adhesion). In various embodiments, the adhesion is the result of injury, surgery (e.g., joint replacement surgery), or inflammation. In some embodiments, the adhesion occurs in a joint, tendon, ligament, eye, abdomen, pericardium, or pelvis. In certain embodiments, the joint is a shoulder joint, knee joint, hip joint, ankle joint, elbow joint, or wrist joint. In alternate embodiments, the adhesion occurs in the abdomen (e.g., an abdominal adhesion) and is further associated with intestinal blockage, bloating, constipation, nausea, vomiting, abdominal pain, or cramps. In further embodiments, the adhesion occurs in the eye (e.g., an eye adhesion) and is further associated with glaucoma.

In a preferred embodiment, the inflammatory condition results from a condition other than bacterial infection. In certain embodiments, the subject has a sterile inflammation.

In some embodiments, the tetracycline (e.g., doxycycline, minocycline, or another tetracycline disclosed herein; preferably doxycycline or sarecycline) is administered orally, transdermally, topically, intravenously, or by injection.

In certain embodiments, a single dose of the tetracycline (e.g., doxycycline, minocycline, or another tetracycline disclosed herein; preferably doxycycline or sarecycline) is administered in an amount between 1 mg and 1000 mg. In preferred embodiments, the dose of the tetracycline administered is an amount between 75 mg and 300 mg, e.g., 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 1 10 mg, 1 15 mg, 120 mg, 125 mg, 130 mg, 135 mg, 140 mg, 145 mg, 150 mg, 155 mg, 160 mg, 165 mg, 170 mg, 175 mg, 180 mg, 185 mg, 190 mg, 195 mg, 200 mg, 205 mg, 210 mg, 215 mg, 220 mg, 225 mg, 230 mg, 235 mg, 240 mg, 245 mg, 250 mg, 255 mg, 260 mg, 265 mg, 270 mg, 275 mg, 280 mg, 285 mg, 290 mg, 295 mg, or 300 mg. In some embodiments, the tetracycline is administered at a submicrobial dose.

In certain embodiments, the tetracycline (e.g., doxycycline, minocycline, or another tetracycline disclosed herein; preferably doxycycline or sarecycline) is administered in intervals of 1 -48 hours, e.g., 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 1 1 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 30 hours, 36 hours, 40 hours, or 48 hours. In preferred embodiments, the tetracycline is administered at least once every 12 or 24 hours. In some embodiments, the tetracycline is administered at least once daily for 1 day to 1 year, e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 1 1 months, or 1 year. In certain embodiments, the tetracycline is administered at least once daily for 1 week to 3 months, e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, or 3 months. In preferred embodiments, the tetracycline is administered at least once daily for 1 week, 1 month (30 days), or 3 months. Preferably the tetracycline is administered for 1 week, 1 month (30 days), or 3 months at a dose in the amount of about 100 mg.

The tetracycline may be selected from the group consisting of doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK-SMA2, tetracycline, chlortetracycline, oxytetracycline, demeclocycline, lymecycline, meclocycline, methacycline, rolitetracycline, clomocycline, metacycline, pipacycline, incyclinide, and a derivative thereof. In preferred embodiments, the tetracycline is doxycycline, minocycline, sarecycline, or omadacycline. In specific embodiments, the tetracycline is doxycycline, minocycline, or sarecycline. In an exemplary embodiment, doxycycline is administered in an amount of 100 mg at least once daily for at least 30 days.

In certain embodiments, the method further involves administering a second therapeutic agent. In particular embodiments, the second therapeutic agent is selected from the group consisting of a nonsteroidal anti-inflammatory drug (NSAID), a COX-2 inhibitor, capsaicin, and gabapentin.

In some embodiments, the tetracycline has not previously been administered to the subject to treat the inflammatory condition.

In other embodiments, the tetracycline has been previously administered to the subject to treat the inflammatory condition.

In a preferred embodiment, the biological sample is or comprises blood.

In some embodiments, the inflammatory biomarker (e.g., a cytokine) is selected from the group consisting of TGF-βΙ , TGF^2, TGF^3, ILI A, IL1 B, TNFa, IL-6, interferon-gamma, IL-8, CX3CL1 , CXCL1 , CXCL10, SDF-1 , IL-2, IL-17, IL-18, IL-23, MMP1 , and MMP3; preferably TGF-βΙ .

In a preferred embodiment, the inflammatory biomarker is TGF-βΙ and the level in the normal subject is <20 ng/ml. In another embodiment, the inflammatory biomarker is IL1 B and the level in the normal subject is <80 pg/ml. In another embodiment, the inflammatory biomarker is TNFa and the level in the normal subject is <10 pg/ml. In another embodiment, the inflammatory biomarker is interferon-gamma and the level in the normal subject is <10 pg/ml. In another embodiment, the inflammatory biomarker is IL1 A and the level in the normal subject is <5 pg/ml. In another embodiment, the inflammatory biomarker is IL-6 and the level in the normal subject is <10 pg/ml. In another embodiment, the inflammatory biomarker is IL-8 and the level in the normal subject is <10 pg/ml. In another embodiment, the inflammatory biomarker is CX3CL1 and the level in the normal subject is <1 ng/ml. In each of the embodiments disclosed above, a level of the noted inflammatory biomarker in a sample from the subject that is equal to or greater than the normal level for the biomarker indicates the presence of an inflammatory condition.

In some embodiments, the inflammatory condition is selected from the group consisting of mTBI, OA, fibromyalgia, diabetic adhesive capsulitis, hypermobility syndrome, exercise intolerance, arthropathy, inflammatory bowel disease, adhesion (e.g., post-operative adhesion, post-operative adhesion after joint replacement surgery, and abdominal adhesion), constipation, diabetes-associated inflammation, Ehlers- Danlos Syndrome, sarcopenia, and liver fibrosis. In preferred embodiments, the inflammatory condition is mTBI, OA, or fibromyalgia.

In some embodiments of any of the methods of the invention, the subject is human.

In an embodiment of any of the methods of the invention, the subject is an athlete, a member of the military, a member of law enforcement, a former athlete, a former member of the military, or a former member of law enforcement. In certain embodiments, the subject is suffering from an inflammatory condition (e.g., mTBI) as a result of an injury (e.g., a head injury and/or combat injury). In a particular embodiment, the subject has suffered multiple injuries (e.g., repetitive head injuries and/or repetitive brain injuries).

In an eighth aspect, the invention features a kit for treating an inflammatory condition, in which the kit includes a device for detecting a level of at least one inflammatory biomarker (e.g., one or more of the inflammatory biomarkers shown in Table 1 , such as TGF-βΙ , TGF^2, TGF^3, MMP1 , or MMP3; preferably TGF-βΙ ) in a biological sample (e.g., blood, cerebrospinal fluid, or synovial fluid), a tetracycline (e.g., doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK-SMA2, another tetracycline described herein, or a derivative thereof; preferably doxycycline or sarecycline), and instructions for use of the tetracycline in a subject in need thereof. In certain embodiments, the inflammatory biomarker is a TGF-β (e.g., TGF-βΙ , TGF^2, or TGF^3).

In some embodiments of the kit, the instructions specify use of an effective amount of the tetracycline in the subject if the level of the at least one inflammatory biomarker is determined, using the device, to be elevated relative to a level of the inflammatory biomarker in a normal subject (e.g., the normal levels of cytokines shown in Table 1 ), or to be elevated relative to a level of the cytokine in a healthy or control subject not having the inflammatory condition. In particular embodiments, the inflammatory biomarker is TGF-βΙ and the elevated level is at least about 20 ng/ml.

In some embodiments of the kit, the inflammatory condition is selected from the group consisting of mTBI, OA, fibromyalgia, diabetic adhesive capsulitis, hypermobility syndrome, exercise intolerance, arthropathy, inflammatory bowel disease, constipation, diabetes-associated inflammation, adhesion (e.g., post-operative adhesion, post-operative adhesion after joint replacement surgery, or abdominal adhesion), Ehlers-Danlos Syndrome, sarcopenia, sarcoidosis, and liver fibrosis. In preferred embodiments, the inflammatory condition is mTBI, OA, fibromyalgia, constipation, or adhesion (e.g., postoperative adhesion, post-operative adhesion after joint replacement surgery, or abdominal adhesion). In various embodiments, the adhesion is the result of injury, surgery (e.g., joint replacement surgery), or inflammation. In some embodiments, the adhesion occurs in a joint, tendon, ligament, eye, abdomen, pericardium, or pelvis. In certain embodiments, the joint is a shoulder joint, knee joint, hip joint, ankle joint, elbow joint, or wrist joint. In alternate embodiments, the adhesion occurs in the abdomen (e.g., an abdominal adhesion) and is further associated with intestinal blockage, bloating, constipation, nausea, vomiting, abdominal pain, or cramps. In further embodiments, the adhesion occurs in the eye (e.g., an eye adhesion) and is further associated with glaucoma.

In certain embodiments of the kit, at least one of the inflammatory biomarkers is selected from the group consisting of TGF-βΙ , TGF^2, TGF^3, ILI A, IL1 B, TNFa, IL-6, interferon-gamma, IL-8, CX3CL1 , CXCL1 , CXCL10, SDF-1 , IL-2, IL-17, IL-18, IL-23, MMP1 , and MMP3.

In a preferred embodiment, the instructions specify that when the inflammatory biomarker is MMP1 , detection of a level of the MMP1 above 30 ng/ml indicates the presence of the inflammatory condition. In a preferred embodiment, the instructions specify that when the inflammatory biomarker is MMP3, detection of a level of the MMP1 above 40 ng/ml indicates the presence of the inflammatory condition. In another embodiment, the instructions specify that when the inflammatory biomarker is IL1 B, detection of a level of the IL1 B above 80 pg/ml indicates the presence of the inflammatory condition. In another embodiment, the instructions specify that when the inflammatory biomarker is TNFa, detection of a level of the TNFa above 10 pg/ml indicates the presence of the inflammatory condition. In another embodiment, the instructions specify that when the inflammatory biomarker is interferon-gamma, detection of a level of the interferon-gamma above 10 pg/ml indicates the presence of the inflammatory condition. In another embodiment, the instructions specify that when the inflammatory biomarker is IL1 A, detection of a level of the IL1 A above 5 pg/ml indicates the presence of the inflammatory condition. In another embodiment, the instructions specify that when the inflammatory biomarker is IL-6, detection of a level of the IL-6 above 10 pg/ml indicates the presence of the inflammatory condition. In another embodiment, the instructions specify that when the inflammatory biomarker is IL-8, detection of a level of the IL-8 above 10 pg/ml indicates the presence of the inflammatory condition. In another embodiment, the instructions specify that when the inflammatory biomarker is CX3CL1 , detection of a level of the CX3CL1 above 1 ng/ml indicates the presence of the inflammatory condition.

In some embodiments of any aspect of the invention, the tetracycline is doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK-SMA2, tetracycline, chlortetracycline, oxytetracycline, demeclocycline, lymecycline, meclocycline, methacycline, rolitetracycline, clomocycline, metacycline, pipacycline, incyclinide, or any derivative thereof (e.g., a minocycline derivative). In preferred embodiments, the tetracycline is doxycycline, minocycline, sarecycline, or omadacycline. In specific embodiments, the tetracycline is doxycycline or sarecycline. Tetracyclines

Tetracyclines are a subclass of polyketides having, e.g., an octahydrotetracene-2-carboxamide skeleton. They are generally derivatives of polycyclic napthacene carboxamide. In some embodiments, the tetracycline is any tetracycline known in the art.

In some embodiments of any of the aspects of the invention, the tetracycline is doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK-SMA2, tetracycline,

chlortetracycline, oxytetracycline, demeclocycline, lymecycline, meclocycline, methacycline,

rolitetracycline, clomocycline, metacycline, pipacycline, incyclinide, or any derivative thereof (e.g., a minocycline derivative). In preferred embodiments, the tetracycline is doxycycline, minocycline, sarecycline, or omadacycline. In specific embodiments, the tetracycline is doxycycline or sarecycline.

Exemplary tetracyclines for use in the methods and kits of the invention include, without limitation, compounds having the structure:

Formula I

wherein R¹ is hydrogen, optionally substituted amino, or halo;

X is CR⁸R⁹ or C=CR¹⁰R^{1 1} ;

R² and R³ are independently hydrogen, hydroxy, or optionally substituted amino;

R⁴ and R⁵ are independently hydrogen, optionally substituted CrC₆ alkyl, or optionally substituted C₂-Cg heterocyclyl CrC₆ alkyl;

R⁶ is hydroxy or -CH₂NR¹²R¹³;

R⁷ is hydrogen, cyano, optionally substituted C Ce alkyl, optionally substituted ( Ce alkynyl, optionally substituted C₆-C₁₀ aryl, or -CH₂NR¹⁴R¹⁵; and

R⁸, R⁹, R¹⁰, R¹¹ , R¹², R¹³, R¹⁴,and R¹⁵ are independently hydrogen, hydroxy, optionally substituted C Ce alkyl, or optionally substituted C Ce alkoxy;

or a pharmaceutically acceptable salt thereof.

In other embodiments tetracyclines include compounds having the structure:

Formula II

wherein R¹ is hydrogen, -N(CH₃)₂, or halo; X is CR⁸R⁹ or C=CR¹⁰R^{1 1} ;

R² and R³ are independently hydrogen, hydroxy, or -N(CH₃)₂;

R is hydrogen, ^**^■ ,

,

CH₃

N

hydroxy o ^H;

R⁷ is hydrogen

and

R⁸, R⁹, R¹⁰, and R^{1 1} are independently hydrogen, hydroxy, or optionally substituted C^Ce alkyl; or a pharmaceutically acceptable salt thereof.

Exemplary tetracyclines include, without limitation:

tetracycline

doxycycline

sarecycline

chlortetracycline

oxytetracycline

demeclocycline

lymecycline

meclocycline

methacycline

rolitetracycline

clomocycline

pipacycline

incvclinide In certain embodiments of the invention, any of the above compounds could be utilized as a tetracycline. Additional tetracyclines that may be utilized according to the methods and kits of the present invention include the compounds (e.g., minocycline derivatives) described in U.S. Patent Publication Nos. 2009/0253660, 2012/0283201 , and 2010/01 13400, each of which is incorporated by reference herein, as well as those described in Higgins et al. 2008 ("Therapeutic Inhibition of Murine Collagen-Induced Arthritis by Non-Antibacterial Derivatives of Minocycline," 15th International Inflammation Research Association Conference, September 21 -24, 2008) and Higgins et al. 2009 ("Inhibition of Murine Collagen-Induced Arthritis by Non-Antibacterial Tetracycline Derivatives," Annual European League Against Rheumatism (EULAR) Congress, June 10-13, 2009), also incorporated by reference herein.

In an embodiment of all aspects of the invention , the biological sample from the subject is, e.g., a biological fluid (e.g., blood, peripheral blood, a blood component (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid (CSF), synovial fluid, or pancreatic fluid), a biological tissue (e.g., chorionic villus sample, muscle, placenta, or dermis), or cells.

Definitions

As used herein, the term "alkyl," "alkenyl" and "alkynyl" include straight-chain, branched-chain and cyclic monovalent substituents, as well as combinations of these, containing only C and H when unsubstituted. Examples include methyl, ethyl, isobutyl, cyclohexyl, cyclopentylethyl, 2-propenyl, 3- butynyl, and the like. The term "cycloalkyl," as used herein, represents a monovalent saturated or unsaturated non-aromatic cyclic alkyl group having between three to nine carbons (e.g., a C3-C9 cycloalkyl), unless otherwise specified, and is exemplified by cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicyclo[2.2.1 .Jheptyl, and the like. When the cycloalkyl group includes one carbon-carbon double bond, the cycloalkyl group can be referred to as a "cycloalkenyl" group. Exemplary cycloalkenyl groups include cyclopentenyl, cyclohexenyl, and the like. When the cycloalkyl group includes one carbon-carbon triple bond, the cycloalkyl group can be referred to as a "cycloalkynyl" group. Exemplary cycloalkynyl groups include cyclopentynyl, cyclohexynyl, and the like.

Typically, the alkyl, alkenyl and alkynyl groups contain 1 -12 carbons (e.g., C1 -C12 alkyl) or 2-12 carbons (e.g., C2-C12 alkenyl or C2-C12 alkynyl). In some embodiments, the alkyl groups are C1 -C8, C1 -C6, C1 -C4, C1 -C3, or C1 -C2 alkyl groups; or C2-C8, C2-C6, C2-C4, or C2-C3 alkenyl or alkynyl groups. Further, any hydrogen atom on one of these groups can be replaced with a substituent as described herein. For example, the term "aminoalkyl" refers to an alkyl group, as defined herein, comprising an optionally substituted amino group (e.g., NH₂).

Heteroalkyl, heteroalkenyl and heteroalkynyl are similarly defined and contain at least one carbon atom but also contain one or more O, S or N heteroatoms or combinations thereof within the backbone residue whereby each heteroatom in the heteroalkyl, heteroalkenyl or heteroalkynyl group replaces one carbon atom of the alkyl, alkenyl or alkynyl group to which the heteroform corresponds. In some embodiments, the heteroalkyl, heteroalkenyl and heteroalkynyl groups have C at each terminus to which the group is attached to other groups, and the heteroatom(s) present are not located at a terminal position. As is understood in the art, these heteroforms do not contain more than three contiguous heteroatoms. In some embodiments, the heteroatom is O or N. The term "heterocyclyl," as used herein represents cyclic heteroalkyi or heteroalkenyl that is, e.g., a 3-, 4-, 5-, 6- or 7-membered ring, unless otherwise specified, containing one, two, three, or four heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. The 5-membered ring has zero to two double bonds, and the 6- and 7-membered rings have zero to three double bonds. The term "heterocyclyl" also represents a heterocyclic compound having a bridged multicyclic structure in which one or more carbons and/or heteroatoms bridges two non-adjacent members of a monocyclic ring, e.g., a quinuclidinyl group. The term "heterocyclyl" includes bicyclic, tricyclic, and tetracyclic groups in which any of the above heterocyclic rings is fused to one, two, or three carbocyclic rings, e.g., an aryl ring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, a cyclopentene ring, or another monocyclic heterocyclic ring, such as indolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, benzofuryl, benzothienyl and the like. Accordingly "heterocyclylalkyl" refers to heterocyclic systems which are coupled to another residue through a carbon chain, saturated or unsaturated, typically of C1 -C8, C1 -C6, or more particularly C1 -C4 or C1 -C3 when saturated or C2-C8, C2-C6, C2-C4, or C2-C3 when unsaturated, including the heteroforms thereof. For greater certainty, heterocyclylalkyl thus includes a heterocyclic group as defined above connected to an alkyl, heteroalkyi, alkenyl, heteroalkenyl, alkynyl or heteroalkynyl moiety also as defined above.

The designated number of carbons in heteroforms of alkyl, alkenyl and alkynyl includes the heteroatom count. For example, if heteroalkyi is defined as C1 -C6, it will contain 1 -6 C, N, O, or S atoms such that the heteroalkyi contains at least one C atom and at least one heteroatom, for example, 1 -5 carbons and 1 N atom, or 1 -4 carbons and 2 N atoms. Similarly, when heteroalkyi is defined as C1 -C6 or C1 -C4, it would contain 1 -5 carbons or 1 -3 carbons respectively, i.e., at least one C is replaced by O, N or S. Accordingly, when heteroalkenyl or heteroalkynyl is defined as C2-C6 (or C2-C4), it would contain 2-6 or 2-4 C, N, O, or S atoms, since the heteroalkenyl or heteroalkynyl contains at least one carbon atom and at least one heteroatom, e.g. 2-5 carbons and 1 N atom, or 2-4 carbons, and 2 O atoms. Further, heteroalkyi, heteroalkenyl or heteroalkynyl substituents may also contain one or more carbonyl groups. Examples of heteroalkyi, heteroalkenyl and heteroalkynyl groups include CH₂OCH₃, CH₂N(CH₃)₂, CH₂OH, (CH₂)_nNR₂, OR, COOR, CONR₂, (CH₂)_nOR,(CH₂)_n COR, (CH₂)_nCOOR, (CH₂)_nSR, (CH₂)_nSOR,

(CH₂)_nS0₂R, (CH₂)_nCONR₂, NRCOR, NRCOOR, OCONR₂, OCOR and the like wherein the R group contains at least one C and the size of the substituent is consistent with the definition of e.g., alkyl, alkenyl, and alkynyl, as described herein (e.g., n is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , or 12).

As used herein, the terms "alkylene," "alkenylene," and "alkynylene," or the prefix "alk" refer to divalent or trivalent groups having a specified size, typically C1 -C2, C1 -C3, C1 -C4, C1 -C6, or C1 -C8 for the saturated groups (e.g., alkylene or alk) and C2-C3, C2-C4, C2-C6, or C2-C8 for the unsaturated groups (e.g., alkenylene or alkynylene). They include straight-chain, branched-chain and cyclic forms as well as combinations of these, containing only C and H when unsubstituted. Because they are divalent, they can link together two parts of a molecule, as exemplified by X in the compounds described herein. Examples are methylene, ethylene, propylene, cyclopropan-1 ,1 -diyl, ethylidene, 2-butene-1 ,4-diyl, and the like. These groups can be substituted by the groups typically suitable as substituents for alkyl, alkenyl and alkynyl groups as set forth herein. Thus C=0 is a C1 alkylene that is substituted by =0, for example. For example, the term "alkaryl," as used herein, represents an aryl group, as defined herein, attached to the parent molecular group through an alkylene group, as defined herein, and the term "alkheteroaryl" refers to a heteroaryl group, as defined herein, attached to the parent molecular group through an alkylene group, as defined herein. The alkylene and the aryl or heteroaryl group are each optionally substituted as described herein.

Heteroalkylene, heteroalkenylene and heteroalkynylene are similarly defined as divalent groups having a specified size, typically C1 -C3, C1 -C4, C1 -C6, or C1 -C8 for the saturated groups and C2-C3, C2-C4, C2-C6, or C2-C8 for the unsaturated groups. They include straight chain, branched chain and cyclic groups as well as combinations of these, and they further contain at least one carbon atom but also contain one or more O, S or N heteroatoms or combinations thereof within the backbone residue, whereby each heteroatom in the heteroalkylene, heteroalkenylene or heteroalkynylene group replaces one carbon atom of the alkylene, alkenylene or alkynylene group to which the heteroform corresponds. As is understood in the art, these heteroforms do not contain more than three contiguous heteroatoms.

The term "alkoxy" represents a chemical substituent of formula -OR, where R is an optionally substituted alkyi group (e.g., C1 -C6 alkyi group), unless otherwise specified. In some embodiments, the alkyi group can be substituted, e.g., the alkoxy group can have 1 , 2, 3, 4, 5 or 6 substituent groups as defined herein.

The term "alkoxyalkyl" represents a heteroalkyl group, as defined herein, that is described as an alkyi group that is substituted with an alkoxy group. Exemplary unsubstituted alkoxyalkyl groups include between 2 to 12 carbons. In some embodiments, the alkyi and the alkoxy each can be further substituted with 1 , 2, 3, or 4 substituent groups as defined herein for the respective group.

The term "amino," as used herein, represents -N(R^N1)₂, wherein each R^N1 is, independently, H, OH, N0₂, N(R^N2)₂, S0₂OR^N2, S0₂R^N2, SOR^N2, an /V-protecting group, alkyi, alkenyl, alkynyl, alkoxy, aryl, alkaryl, cycloalkyl, alkcycloalkyl, heterocyclyl (e.g., heteroaryl), alkheterocyclyl (e.g., alkheteroaryl), or two R^N1 combine to form a heterocyclyl or an /V-protecting group, and wherein each R^N2 is, independently, H, alkyi, or aryl. In a preferred embodiment, amino is -NH₂, or -NHR^N1 , wherein R^N1 is, independently, OH, N0₂, NH₂, NR^N2 ₂, S0₂OR^N2, S0₂R^N2, SOR^N2, alkyi, or aryl, and each R^N2 can be H, alkyi, or aryl. The term "aminoalkyl," as used herein, represents a heteroalkyl group, as defined hrein, that is described as an alkyi group, as defined herein, substituted by an amino group, as defined herein. The alkyi and amino each can be further substituted with 1 , 2, 3, or 4 substituent groups as described herein for the respective group. For example, the alkyi moiety may comprise an oxo (=0) substituent.

"Aromatic" moiety or "aryl" moiety refers to any monocyclic or fused ring bicyclic system which has the characteristics of aromaticity in terms of electron distribution throughout the ring system and includes a monocyclic or fused bicyclic moiety such as phenyl or naphthyl; "heteroaromatic" or

"heteroaryl" also refers to such monocyclic or fused bicyclic ring systems containing one or more heteroatoms selected from O, S and N. The inclusion of a heteroatom permits inclusion of 5 membered rings to be considered aromatic as well as 6 membered rings. Thus, typical aromatic/heteroaromatic systems include pyridyl, pyrimidyl, indolyl, benzimidazolyl, benzotriazolyl, isoquinolyl, quinolyl, benzothiazolyl, benzofuranyl, thienyl, furyl, pyrrolyl, thiazolyl, oxazolyl, isoxazolyl, benzoxazolyl, benzoisoxazolyl, imidazolyl and the like. Because tautomers are theoretically possible, phthalimido is also considered aromatic. Typically, the ring systems contain 5 12 ring member atoms or 6-10 ring member atoms. In some embodiments, the aromatic or heteroaromatic moiety is a 6-membered aromatic rings system optionally containing 1 -2 nitrogen atoms. More particularly, the moiety is an optionally substituted phenyl, pyridyl, indolyl, pyrimidyl, pyridazinyl, benzothiazolyl, benzimidazolyl, pyrazolyl, imidazolyl, isoxazolyl, thiazolyl, benzothiazolyl, indolyl, or imidazopyridinyl. Even more particularly, such moiety is phenyl, pyridyl, thiazolyl, imidazopyridinyl, or pyrimidyl and even more particularly, it is phenyl.

"O-aryl" or "O-heteroaryl" refers to aromatic or heteroaromatic systems which are coupled to another residue through an oxygen atom. A typical example of an O-aryl is phenoxy. Similarly, "arylalkyi" refers to aromatic and heteroaromatic systems which are coupled to another residue through a carbon chain, saturated or unsaturated, typically of C1 -C8, C1 -C6, or more particularly C1 -C4 or C1 -C3 when saturated or C2-C8, C2-C6, C2-C4, or C2-C3 when unsaturated, including the heteroforms thereof. For greater certainty, arylalkyi thus includes an aryl or heteroaryl group as defined above connected to an alkyl, heteroalkyl, alkenyl, heteroalkenyl, alkynyl or heteroalkynyl moiety also as defined above. Typical arylalkyls would be an aryl(C6-C12)alkyl(C1 -C8), aryl(C6-C12)alkenyl(C2-C8), or aryl(C6-C12)alkynyl(C2- C8), plus the heteroforms. A typical example is phenylmethyl, commonly referred to as benzyl.

Halo may be any halogen atom, especially F, CI, Br, or I, and more particularly it is fluoro or chloro.

The term "haloalkyl," as used herein, represents an alkyl group, as defined herein, substituted by a halogen group (i.e., F, CI, Br, or I). A haloalkyl may be substituted with one, two, three, or, in the case of alkyl groups of two carbons or more, four halogens. Haloalkyl groups include perfluoroalkyls. In some embodiments, the haloalkyl group can be further substituted with 1 , 2, 3, or 4 substituent groups as described herein for alkyl groups.

The term "hydroxy," as used herein, represents an -OH group.

The term "hydroxyalkyl," as used herein, represents an alkyl group, as defined herein, substituted by one to three hydroxy groups, with the proviso that no more than one hydroxy group may be attached to a single carbon atom of the alkyl group, and is exemplified by hydroxymethyl, dihydroxypropyl, and the like.

The term "/V-protecting group," as used herein, represents those groups intended to protect an amino group against undesirable reactions during synthetic procedures. Commonly used /V-protecting groups are disclosed in Greene, "Protective Groups in Organic Synthesis," 3^rd Edition (John Wiley & Sons, New York, 1999), which is incorporated herein by reference. /V-protecting groups include acyl, aryloyl, or carbamyl groups such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2- bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, a-chlorobutyryl, benzoyl, 4- chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and chiral auxiliaries such as protected or unprotected D, L or D, L-amino acids such as alanine, leucine, phenylalanine, and the like; sulfonyl-containing groups such as benzenesulfonyl, p-toluenesulfonyl, and the like; carbamate forming groups such as

benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5- dimethoxybenzyl oxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro- 4,5-dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, 1 -(p-biphenylyl)-l - methylethoxycarbonyl, a,a-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxy carbonyl, t- butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2,2,2, -trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxy carbonyl, fluorenyl-9- methoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl,

phenylthiocarbonyl, and the like, alkaryl groups such as benzyl, triphenylmethyl, benzyloxymethyl, and the like and silyl groups such as trimethylsilyl, and the like. Preferred /V-protecting groups are formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, alanyl, phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc), and benzyloxycarbonyl (Cbz).

An "oxo" group is a substituent having the structure C=0, where there is a double bond between a carbon and an oxygen atom.

A "thiol" group is a substituent have the structure -S-H.

Typical optional substituents on aromatic or heteroaromatic groups include independently halo, CN, N0₂, CF₃, OCF₃, COOR', CONR'₂, OR', SR", SOFT, S0₂R', NR'₂, NR'(CO)R',NR'C(0)OR',

NR'C(0)NR'₂, NR'S0₂NR'₂, or NR'S0₂R', wherein each R' is independently H or an optionally substituted group selected from alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, and aryl (all as defined above); or the substituent may be an optionally substituted group selected from alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, aryl, heteroaryl, O-aryl, O-heteroaryl and arylalkyl.

Optional substituents on a non-aromatic group (e.g., alkyl, alkenyl, and alkynyl groups), are typically selected from the same list of substituents suitable for aromatic or heteroaromatic groups, except as noted otherwise herein. A non-aromatic group may also include a substituent selected from =0 and =NOR' where R' is H or an optionally substituted group selected from alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteralkynyl, heteroaryl, and aryl (all as defined above).

In general, a substituent group (e.g., alkyl, alkenyl, alkynyl, or aryl (including all heteroforms defined above) may itself optionally be substituted by additional substituents. The nature of these substituents is similar to those recited with regard to the substituents on the basic structures above. Thus, where an embodiment of a substituent is alkyl, this alkyl may optionally be substituted by the remaining substituents listed as substituents where this makes chemical sense, and where this does not undermine the size limit of alkyl per se; e.g., alkyl substituted by alkyl or by alkenyl would simply extend the upper limit of carbon atoms for these embodiments, and is not included. However, alkyl substituted by aryl, amino, halo and the like would be included. For example, where a group is substituted, the group may be substituted with 1 , 2, 3, 4, 5, or 6 substituents. Optional substituents include, but are not limited to: C1 -C6 alkyl or heteroaryl, C2-C6 alkenyl or heteroalkenyl, C2-C6 alkynyl or heteroalkynyl, halogen; aryl, heteroaryl, azido(-N₃), nitro (-N0₂), cyano (-CN), acyloxy (OC(=0)R'), acyl (-C(=0)R'), alkoxy (-OR'), amido (-NR'C(=0)R" or -C(=0)NRR'), amino (NRR'), carboxylic acid (-C0₂H), carboxylic ester (-C0₂R'), carbamoyl (OC(=0)NR'R" or -NRC(=0)OR'), hydroxy (OH), isocyano (-NC), sulfonate (S(=0)₂OR), sulfonamide (S(=0)₂NRR' or -NRS(=0)₂R'), or sulfonyl (S(=0)₂R), where each R or R' is selected, independently, from H, C1 -C6 alkyl or heteroaryl, C2-C6 alkenyl or heteroalkenyl, 2C-6C alkynyl or heteroalkynyl, aryl, or heteroaryl. A substituted group may have, for example, 1 , 2, 3, 4, 5, 6, 7, 8, or 9 substituents.

In some embodiments, the invention features moieties that are amino acid residues. The amino acid residue may be of a naturally occurring amino acid (e.g., Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val), or the amino acid residue may be of a non- naturally occurring amino acid. A "non-naturally occurring amino acid" is an amino acid which is not naturally produced or found in a mammal. Examples of non-naturally occurring amino acids include D- amino acids; an amino acid having an acetylaminomethyl group attached to a sulfur atom of a cysteine; a pegylated amino acid; the omega amino acids of the formula NH₂(CH₂)_nCOOH wherein n is 2-6, neutral nonpolar amino acids, such as sarcosine, t-butyl alanine, t-butyl glycine, N-methyl isoleucine, and norleucine; phenylglycine; citrulline; methionine sulfoxide; cysteic acid; ornithine; and hydroxyproline.

The term "inflammatory condition," as used herein, refers to any disease, disorder, or condition associated with an inflammatory response. The term "inflammatory response" encompasses a broad variety of biological activities, including, for example, pain, heat, redness, swelling, and loss of function. Inflammatory conditions may include, but are not limited to, mTBI, OA, fibromyalgia, diabetic adhesive capsulitis, hypermobility syndrome, exercise intolerance, arthropathy, inflammatory bowel disease, constipation, diabetes-associated inflammation, adhesion (e.g., post-operative adhesion, post-operative adhesion after joint replacement surgery, or abdominal adhesion), Ehlers-Danlos Syndrome, sarcopenia, sarcoidosis, and liver fibrosis. An adhesion can be the result of injury, surgery (e.g., joint replacement surgery), or inflammation. An adhesion can occur, for example, in a joint, tendon, ligament, eye, abdomen, pericardium, or pelvis. The joint can be, e.g., a shoulder joint, knee joint, hip joint, ankle joint, elbow joint, or wrist joint. Alternatively, the adhesion can occur, e.g., in the abdomen and be further associated with intestinal blockage, bloating, constipation, nausea, vomiting, abdominal pain, or cramps. The adhesion can also occur, e.g., in the eye and be further associated with glaucoma. An inflammatory condition may be associated with fibrosis or a fibrotic condition.

By "inflammatory condition results from a condition other than bacterial infection," "inflammatory condition resulting from a condition other than bacterial infection," "inflammatory condition results from a cause other than bacterial infection," or variants thereof is meant any indication or condition resulting in symptoms of inflammation in which the cause of the symptoms of inflammation is not bacterial infection. Such inflammatory conditions may include, for example, localized inflammation, systemic inflammation, and/or sterile inflammation. In some embodiments, a subject to be treated according to the methods of the invention or using the kits of the invention also does not have inflammation caused by a non-bacterial infective organism (e.g., a fungus, virus, or protist).

By "sterile inflammation" or "sterile inflammatory condition" is meant an inflammation that is not caused by infection with a pathogen, such as a bacterium, virus, protist, or fungus. Causes of sterile inflammation include, for example, physical trauma (e.g., head trauma) or mitochondrial nucleic acid released from cells as a result of trauma. A subject may have an inflammation that has both "infective" and "sterile" etiologies. A sterile inflammation may be indicated by an increased level of a mitochondrial nucleic acid (cytochrome B mitochondrial nucleic acid) of > 1 g/ mL or > 0.5 pg/ mL in the blood of the subject in the absence of, or a low level of, microbial (e.g., bacterial) nucleic acid in the subject's blood (see PCT Pub. No. WO 201 1/069058, incorporated herein in its entirety). Sterile inflammatory conditions include, but are not limited to, mTBI, OA, fibromyalgia, diabetic adhesive capsulitis, hypermobility syndrome, exercise intolerance, arthropathy, inflammatory bowel disease, constipation, diabetes- associated inflammation, adhesion (e.g., an adhesion as described herein), Ehlers-Danlos Syndrome, sarcopenia, sarcoidosis, and liver fibrosis.

By "symptoms of inflammation" is meant one or more (e.g., two, three, or four) physical manifestations of an inflammatory response (e.g., a sterile inflammation or an infective inflammation). Non-limiting examples of symptoms of inflammation include: altered body temperature (e.g., less than

36°C or greater than 38°C), increased heart rate (e.g., greater than 90 beats per minute), tachypnea (e.g., greater than 20 breaths per minute), decreased arterial pressure of C0₂ (e.g., less than 4.3 kPa), altered white blood count (e.g., less than 4,000 cells/mm³ or greater than 12,000 cells/mm³), increased histamine levels (e.g., greater than 60 ng/niL in blood), increased leukotriene B4 levels (e.g., greater than 30 pg/mL or greater than 35 pg/mL in blood), increased prostaglandin levels (e.g., greater than 3.0 ng mL in blood), increased levels of pro-inflammatory biomarkers (e.g., increased levels of pro-inflammatory cytokines, for example, greater than 20 ng mL TNF-a and/or greater than 10 pg/mL IL-6), redness, soreness, pain, swelling, or combinations thereof.

By the term "diabetes-associated inflammation" is meant inflammation, an inflammatory condition, and/or symptoms of inflammation associated with, e.g., type 1 diabetes or type 2 diabetes. Diabetes- associated inflammation may be age-related, and may further involve extracellular matrix crosslinking and/or fibrosis in tissues such as adipose tissue, the liver, pancreatic islets, and the vasculature. For example, collagen cross-linking in the form of glucosepane is known to be associated with diabetes and aging (Monnier et al., Ann. N. Y. Acad. Sci. 1043: 533-544, 2005).

"Conscious," as used herein, has the conventional meaning, as set forth in Plum et al, The Diagnosis of Stupor and Coma, CNS Series, Philadelphia:Davis (1982), which is hereby incorporated by reference. Conscious subjects include those who have a capacity for reliable, reproducible, interactive behavior evidencing awareness of self or the environment. Conscious subjects include subjects who recover consciousness with less severe brain injury but who, because of their impaired cognitive function, do not reach independent living. Conscious subjects do not include those who exhibit wakefulness but lack interaction (e.g., those deemed to be in a persistent vegetative state).

By the terms "biological sample," "body fluid sample," or "sample," as used herein, is meant any specimen (e.g., blood, peripheral blood, blood component (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid (CSF), synovial fluid, tissue (e.g., placental, dermal, or muscle), pancreatic fluid, chorionic villus sample, and cells) taken from a subject. Preferably, the sample is blood, peripheral blood, or a blood component (e.g., serum or plasma). A biological sample can be obtained by methods well known in the art. For example, samples from a subject may be obtained by venipuncture, resection, bronchoscopy, fine needle aspiration, bronchial brushings, or from sputum, pleural fluid, urine or blood, such as serum or plasma. Genes or gene products, such as mRNA, cDNA, or protein (e.g., an inflammatory biomarker such as those described in Table 1 ), can be detected from these samples.

Furthermore, the genes or gene products may be extracted from the biological sample prior to analysis, which may permit detection of such genes or gene products at higher precision, accuracy, or sensitivity. For example, an inflammatory biomarker, such as a cytokine (e.g., a TGF-β), may be extracted from blood plasma as described by Anscher et al. (New England J. Med. 328(22): 1592-1598, 1993). By screening such biological samples, a simple early diagnosis or differential diagnosis can be achieved. In addition, the progress of therapy can be monitored more easily by testing such biological samples for target genes or gene products. Furthermore, the prediction of outcome or response to therapy can be tested more easily by testing such biological samples for target genes or gene products.

The terms "biomarker" or "inflammatory biomarker," as used herein, refer to a polynucleotide or polypeptide, the detection of which is desired. Exemplary biomarkers include, but are not limited to, the inflammatory biomarkers (e.g., cytokines) listed in Table 1 (e.g., TGF-βΙ , TGF^2, or TGF^3). By "TGF-β" is meant a member of the TGF-β gene family or a polypeptide encoded by a gene in the TGF-β family. Members of the TGF-β family include, for example, TGF-βΙ , TGF^2, and TGF^3.

The term "about," as used herein, means that a particular value is approximate to, near to, or close to a defined value or range, for example, within 10% above or below (e.g., within +1 %, +2%, +3%, +4%, +5%, +6%, +7%, +8%, +9%, or +10%) the defined value or range.

As used herein, and as well understood in the art, "to treat" a condition, "treatment" of the condition (e.g., the conditions described herein, such as inflammatory conditions), or "therapy" is an approach for obtaining beneficial or desired results, such as clinical results, and can be performed either for prophylaxis or during the course of clinical pathology. Beneficial or desired results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions; diminishment of extent of disease, disorder, or condition; stabilized (i.e., not worsening) state of disease, disorder, or condition; preventing spread of disease, disorder, or condition; delay or slowing the progress of the disease, disorder, or condition; amelioration or palliation of the disease, disorder, or condition; and remission (whether partial or total), whether detectable or undetectable. "Palliating" a disease, disorder, or condition means that the extent and/or undesirable clinical manifestations of the disease, disorder, or condition are lessened and/or time course of the progression is slowed or lengthened, as compared to the extent or time course in the absence of treatment.

The term an "effective amount" of an agent (e.g., doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK-SMA2, another tetracycline described herein, or a derivative thereof; preferably doxycycline or sarecycline) refers to that amount sufficient to effect beneficial or desired results, such as clinical results, and, as such, an "effective amount" depends upon the context in which it is being applied, but which can be determined according to known methods and techniques by one skilled in the art or based on the guidance disclosed herein.

As used herein, the term "submicrobial dose" refers to an amount of a tetracycline (e.g., doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK-SMA2, another tetracycline described herein, or a derivative thereof; preferably doxycycline or sarecycline) that will not have significant antimicrobial activity (e.g., antibacterial, antiviral, or antifungal activities). "Significant antimicrobial activity" refers to clinically relevant antimicrobial activity. Submicrobial doses include, for example, doses below 200 mg/day, doses below 100 mg/ day or doses below 50 mg/ day. Exemplary submicrobial doses include doses of 5 mg/day, 10 mg/day, 15 mg/day, 20 mg/day, 25 mg/day, 30 mg/ day, 40 mg/day, 50 mg/day, 60 mg/day, 70 mg/day, 80 mg/day, 90 mg/day, 100 mg/ day, 125 mg/day, 150 mg/day, or 175 mg/day. For example, a preferred submicrobial dose of doxycycline is 100 mg/day. A daily submicrobial dose may be administered all at once (e.g., 100 mg administered once daily) or in multiple administrations over the course of a day (e.g., 20 mg administered five times over the course of a day).

By "course of therapy" is meant the length of time from the initiation of a therapy until the conclusion of the therapy.

As used herein, the terms "subject" or "patient" refer to any organism to which a compound or composition in accordance with the invention (e.g., a tetracycline, such as doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK-SMA2, another tetracycline described herein, or a derivative thereof; preferably doxycycline or sarecycline) may be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. A subject to be treated with a compound or composition described herein may be one who has been diagnosed by a medical practitioner as having an inflammatory condition described herein (e.g., mTBI, OA, fibromyalgia, diabetic adhesive capsulitis, hypermobility syndrome, exercise intolerance, arthropathy, inflammatory bowel disease, constipation, diabetes-associated inflammation, adhesion (e.g., an adhesion as described herein), Ehlers-Danlos Syndrome, sarcopenia, sarcoidosis, or liver fibrosis), or one at risk for developing such an inflammatory condition. Diagnosis may be performed by any technique or method known in the art. One skilled in the art will understand that a subject may have been diagnosed as having the inflammatory condition using a standard test or examination or may have been identified, without examination, as one at high risk due to the presence of one or more risk factors. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans; preferably the subject is a human).

By "elevated" or "elevated level" is meant that the expression level of a biomarker (e.g., an inflammatory biomarker such as a TGF-β) in a subject is higher than the expression level of the same biomarker in, for example, a control (e.g., a normal subject, the median expression level in a population of normal subjects, or the median expression level in the overall population). An elevated level of a biomarker in a subject may be, e.g., statistically significantly higher than that of the control (e.g., as determined using a statistical test as well known in the art, e.g., a t-test).

The term "normal subject," as used herein, refers to a subject lacking an inflammation and/or an inflammatory condition (e.g., mTBI, OA, fibromyalgia, diabetic adhesive capsulitis, hypermobility syndrome, exercise intolerance, arthropathy, inflammatory bowel disease, constipation, diabetes- associated inflammation, adhesion, Ehlers-Danlos Syndrome, sarcopenia, sarcoidosis, or liver fibrosis). A normal subject may be one in which a level of an inflammatory biomarker, e.g., one or more of the inflammatory biomarkers listed in Table 1 (e.g., TGF-βΙ , TGF^2, or TGF^3; preferably TGF-βΙ ), is within the "normal level."

Detailed Description of the Invention

The present invention features methods and kits for treating subjects for inflammatory conditions (e.g., subjects having an elevated level of at least one TGF-β relative to a level in a normal subject). In particular, the invention may be useful for treating subjects with inflammatory conditions resulting from a condition other than bacterial infection, including, but not limited to, sterile inflammation, mTBI, OA, fibromyalgia, diabetic adhesive capsulitis, hypermobility syndrome, exercise intolerance, arthropathy, inflammatory bowel disease, constipation, diabetes-associated inflammation, post-operative adhesion, post-operative adhesion after joint replacement surgery, abdominal adhesion, Ehlers-Danlos Syndrome, sarcopenia, sarcoidosis, and liver fibrosis. Preferably, an inflammatory condition that can be treated according to the present invention is selected from mTBI, OA, and fibromyalgia. Methods and kits of the invention can be used to determine if the subject has such an inflammatory condition and for

administering an effective amount of a tetracycline compound (e.g., doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK-SMA2, another tetracycline described herein, or a derivative thereof; preferably doxycycline or sarecycline) to the subject. The inventor has discovered that inflammatory conditions (e.g., those caused by head trauma, such as mTBI, or other sterile inflammatory conditions) may modulate the expression or activity of inflammatory biomarkers (e.g., cytokines, such as TGF-βΙ ), and that this modulation may be indicative of such inflammatory conditions. Methods for detecting an inflammatory condition in a subject are thus contemplated, involving, for example, the determination of a level of at least one inflammatory biomarker (e.g., a cytokine, such as TGF-βΙ ) in a biological sample obtained from the subject (e.g., blood or cerebrospinal fluid).

Furthermore, the invention includes treating subjects with such inflammatory conditions with an effective amount of a tetracycline compound (e.g., doxycycline, minocycline, sarecycline, omadacycline, PTK- RA01 , PTK-MS01 , PTK-SMA2, another tetracycline described herein, or a derivative thereof; preferably doxycycline or sarecycline). Furthermore, the efficacy of such treatments may be monitored by determining a level of an inflammatory biomarker (e.g., a cytokine, such as TGF-βΙ ) in the subject during and/or following therapy.

The invention features methods of treating subjects, involving determining if the subject has an inflammatory condition (e.g., mTBI, OA, fibromyalgia, diabetic adhesive capsulitis, hypermobility syndrome, exercise intolerance, arthropathy, inflammatory bowel disease, constipation, diabetes- associated inflammation, adhesion (e.g., post-operative adhesion, post-operative adhesion after joint replacement surgery, or abdominal adhesion), Ehlers-Danlos Syndrome, sarcopenia, sarcoidosis, and liver fibrosis; preferably mTBI, OA, or fibromyalgia), and if so, administering an effective amount of a tetracycline (e.g., doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK- SMA2, another tetracycline described herein, or a derivative thereof; preferably doxycycline or sarecycline) to the subject. Such an inflammatory condition may not be the result of a bacterial infection (e.g., the subject may have sterile inflammation). The tetracycline may be delivered to the subject via various routes of administration (e.g., oral, topical, or transdermal administration) and may be formulated in a pharmaceutical composition as the sole active ingredient or in combination with one or more additional therapies or therapeutic agents (e.g. a non-steroidal anti-inflammatory drug (NSAID), a COX-2 inhibitor, capsaicin, or gabapentin). Additional therapies may include, for example, physical therapy (e.g., eccentric resistance training).

Also contemplated are methods of diagnosing a subject for an inflammatory condition or detecting the presence of an inflammatory condition in a subject, by determining a level of at least one inflammatory biomarker, such as the inflammatory biomarkers shown in Table 1 , in a biological sample obtained from the subject, such as a blood sample. A determination that the level of the inflammatory biomarker(s) in the biological sample is elevated relative to a level in a normal subject indicates that the subject has an inflammatory condition and/or may benefit from treatment with a tetracycline of the invention (e.g., doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK-SMA2, another tetracycline described herein, or a derivative thereof; preferably doxycycline or sarecycline). If the subject is found to have an inflammatory condition or to be in need of treatment using the above method, an effective amount of a tetracycline may be administered to the subject based on the level of the cytokine(s) in the biological sample. For example, if the biological sample is found to have elevated levels of the inflammatory biomarker, an elevated amount of the tetracycline may be administered to the subject.

The invention further features kits for treating inflammatory conditions. Kits contemplated herein include a device for detecting a level of at least one inflammatory biomarker (e.g., an inflammatory cytokine), such as those shown in Table 1 , in a biological sample, a tetracycline, and instructions for administering the tetracycline to a subject in need thereof. In preferred embodiments, an effective amount of the tetracycline will be administered to the subject if the level of the one or more inflammatory biomarkers is determined, using the device or otherwise, to be elevated relative to a level of that inflammatory biomarker in a normal subject. Methods for diagnosing an inflammatory condition

The present invention features methods and kits for detecting the presence of an inflammatory condition in a subject. Such inflammatory conditions include, without limitation, mTBI, OA, fibromyalgia, diabetic adhesive capsulitis, hypermobility syndrome, exercise intolerance, arthropathy, inflammatory bowel disease, constipation, diabetes-associated inflammation, adhesion (e.g., post-operative adhesion, post-operative adhesion after joint replacement surgery, and abdominal adhesion), Ehlers-Danlos Syndrome, sarcopenia, sarcoidosis, and liver fibrosis. An adhesion can be the result of, e.g., injury, surgery, or inflammation. An adhesion resulting from surgery can be, for example, post-operative adhesion after joint replacement surgery. Adhesion can occur, e.g., in a joint, tendon, ligament, eye, abdomen, pericardium, or pelvis. Joints that can be affected by adhesion include, for example, a shoulder joint, knee joint, hip joint, ankle joint, elbow joint, or wrist joint. An adhesion occurring in the abdomen can be further associated with, e.g., intestinal blockage, bloating, constipation, nausea, vomiting, abdominal pain, or cramps. An adhesion occurring in the eye can be further associated with glaucoma. In preferred embodiments, the inflammatory conditions may be mTBI, OA, fibromyalgia, constipation, or an adhesion as described herein. Each of these inflammatory conditions can be diagnosed according to accepted guidelines, methods, and protocols known in the art. Such diagnostic methods may be used to detect the presence of an inflammatory condition in a subject or to monitor the efficacy of a treatment (e.g., treatment with doxycycline, minocycline, sarecycline, omadacycline, PTK- RA01 , PTK-MS01 , PTK-SMA2, another tetracycline described herein, or a derivative thereof; preferably doxycycline or sarecycline), and/or can be combined with methods of the present invention to determine the presence of an inflammatory condition in a subject. Indicia of these conditions are discussed below.

Methods of diagnosing an inflammatory condition described herein, or monitoring the efficacy of a treatment (e.g., treatment with doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK- MS01 , PTK-SMA2, another tetracycline described herein, or a derivative thereof; preferably doxycycline or sarecycline) for such an inflammatory condition, may involve detecting a difference in a biological activity, process, or structure in vitro or in vivo, including but not limited to fibrosis, cell growth, cell proliferation, apoptosis, cell migration, cell differentiation, cell morphology, blood vessel growth or maturation, tissue inflammation (e.g., symptoms such as localized pain, redness, or swelling), the presence or absence of sarcopenia, developmental phenotype, or any other biological activity known in the art. Preferably, the diagnostic method may involve detecting fibrosis in a tissue or organ affected by an inflammatory condition (e.g., joint fibrosis, synovial fibrosis, muscle fibrosis, or liver fibrosis). Such diagnostic methods can be combined with analysis of inflammatory biomarkers, e.g., according to the methods described herein.

Mild traumatic brain injury (mTBI)

The invention features methods for treating a subject diagnosed with an inflammatory condition, such as mTBI, with a tetracycline (e.g., doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK-SMA2, another tetracycline described herein, or a derivative thereof; preferably doxycycline or sarecycline). Subjects may be diagnosed with mTBI according to known and accepted guidelines, methods, or protocols, or by using the methods and kits described herein for detecting the presence of mTBI in a subject. Diagnostic methods that can be used to determine whether a subject (e.g., a mammal, such as a human) exposed to head trauma has mTBI may include evaluating whether the subject exhibits one or more of the following conditions: memory loss; pupil dilation; convulsions; distorted facial features; fluid draining from nose, mouth, or ears; fracture in the skull or face; bruising of the face; swelling at the site of injury; scalp wound; impaired hearing, smell, taste, or vision; inability to move one or more limbs; irritability; personality changes; unusual behavior; confusion; drowsiness; low breathing rate; drop in blood pressure; restlessness, clumsiness; lack of coordination, severe headache, slurred speech; stiff neck; and vomiting. Preferably, the subject is conscious or unconscious but not comatose. A mild brain injury that occurs without loss of consciousness may leave a subject with merely a dazed feeling or confused state lasting a short time.

mTBI may also be diagnosed by determining the presence of or level of a particular biomarker in a body fluid sample (e.g., blood) obtained from the selected subject. Presence of the biomarker or increased levels of the biomarker in the body fluid sample, relative to a standard or control, indicates that the subject has suffered mTBI. mTBI may also by indicated by any one or more of the following:

cognition impairment; language impairment; conduct disorder; motor disorder; and any other neurological dysfunction. mTBI may occur with no loss of consciousness and possibly only a dazed feeling or confused state lasting a short time. The subject who is exposed to the head trauma may exhibit extracranial injuries or may exhibit no extra-cranial injuries. The head trauma may be produced, at least in part, by brain injuries including those produced by blunt head trauma or missile penetration (entry of an object going through the skull). A subject who is conscious after exposure to a head trauma may be asymptomatic or lack any visible symptoms of traumatic brain injury. Conversely, a conscious subject may exhibit various symptoms of brain injury and cognitive dysfunction. A subject who is unconscious at the time of injury may present with symptoms such as a concussion or intracranial hemorrhage (e.g. intra- axial hematoma, epidural hematoma, and subdural hematoma).

As indicated above, the subject exposed to head trauma may exhibit extracranial injuries.

Exemplary extra-cranial injuries include open head injuries, such as a visible assault to the head. Extra- cranial injuries may result from a gunshot wound, an accident or an object going through the skull into the brain ("missile injury to the brain"). This type of brain injury is likely to damage a specific area of the brain. Alternatively, the subject exposed to a head trauma may exhibit only superficial external injuries or no extra-cranial injuries. In this instance, the subject may have no visible injury (e.g. a closed head injury), or may exhibit those symptoms by deficits in attention, intention, working memory, and/or awareness as described herein.

A brain injury such as mTBI may occur when there is a blow to the head as in a motor vehicle accident, a fall, or a concussive blast. The brain, which is inside the skull, turns and twists on its axis (the brain stem), causing localized or widespread damage. Also, the brain, a soft mass surrounded by fluid that allows it to "float," may rebound against the skull resulting in further damage. In response to the head trauma, changes occur in the brain, which require monitoring to prevent further damage. The brain's size frequently increases after a severe head injury. This is called brain swelling and occurs when there is an increase in the amount of blood to the brain. Later in the illness, water may collect in the brain, which is called brain edema. Both brain swelling and brain edema result in excessive pressure in the brain called intracranial pressure ("ICP").

mTBI may result in persisting debility, such as post-traumatic epilepsy, persistent vegetative state, or post-traumatic dementia in the absence of proper treatment. Other complications and late effects of brain injury include, but are not limited to, coma, meningitis, post-traumatic epilepsy, post-traumatic dementia, degeneration of nerve fibers, post-traumatic syringomyelia, or hemorrhage, for example.

Although medical care administered may be minimal in the context of mTBI, persons with brain injury without coma may experience symptoms and impairments similar to those suffered by the survivor of a severe brain injury.

The determination of whether the subject has suffered mTBI can be completed immediately following a head trauma, or at any time thereafter.

Brain injury symptoms frequently manifest themselves in combined deficits of attention, intention, working memory, and/or awareness. As used herein, attention refers to the cognitive function that provides the capacities for selection of internal or external stimuli and thoughts, supports the preparation of intended behaviors (e.g., speeds perceptual judgments and reaction times), and supports the maintenance of sustained cognition or motor behaviors (e.g., the focusing of attention). Intention, as used herein, refers to the mechanism of response failures (i.e., lack of behavioral interaction) which is not due to a perceptual loss (i.e., intention is the cognitive drive linking sensory- motor integration to behavior). Intention deficits include failure to move a body part despite intact motor pathways, awareness, and sensory processing as demonstrated by neurophysiological and neuropsychological evaluation. Another example of a patient's intention deficit is a failure to initiate action of any kind despite evidence of awareness or action produced by stimulation. Loss of intention is a disorder of cognitive function, as defined herein, and is a major division of the neuropsychological disorder of neglect, which may be present in many patients with cognitive loss following brain injury caused by a head trauma. Working memory, as used herein, refers to the fast memory process required for on-line storage and retrieval of information, including processes of holding incoming information in short-term memory before it can be converted into long-term memory and processes which support the retrieval of established long-term (episodic) memories. Deficits in awareness relate to impaired perceptual awareness, as described above. Clinical signs of these brain injuries also include profound hemi-spatial neglect, disorders of motor intention, disorders of impaired awareness of behavioral control, or apathy and cognitive slowing.

Osteoarthritis

The invention features methods for treating a subject diagnosed with an inflammatory condition, such as osteoarthritis (OA), with a tetracycline (e.g., doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK-SMA2, another tetracycline described herein, or a derivative thereof;

preferably doxycycline or sarecycline). Subjects may be diagnosed with OA according to known and accepted guidelines, methods, or protocols, or by using the methods and kits described herein to detect the presence of OA in a subject. Techniques available for diagnosing OA include general observation of the patient, magnetic resonance imaging (MRI), and x-radio graphic methods, such as by observation of joint space narrowing. One of the important pathological features of OA is the progressive degradation of articular cartilage, which is an avascular and aneural tissue consisting of an extracellular matrix (ECM), tissue fluid and chondrocytes as a single cell type. The ECM consists of a network of collagen (collagen II, IX and XI) and proteoglycans (mainly aggrecan) that together determine the physical and mechanical properties of cartilage. The cartilage damage occurs due to mechanical stress on the joints and the enzymatic activity of metalloproteinases (e.g., MMPs-1 . -2, -3, -13), and aggrecanases (ADAMTS-4 and - 5) on the ECM, that are induced by the activity of pro- inflammatory biomarkers (e.g., pro-inflammatory cytokines), such as IL-Ιβ and TNF-a.

Formal diagnostic criteria are often used to diagnose OA. OA of the knee is diagnosed by the presence of knee pain plus at least three of the following characteristics: age greater than 50 years, morning stiffness lasting less than 30 minutes, crackling or grating sensation (crepitus), bony tenderness of the knee, bony enlargement of the knee, no detectable warmth of the joint to the touch. Laboratory tests, including, but not limited to, complete blood counts, urinanalysis, rheumatoid factor tests, and/or x- rays are often used in addition to these criteria. Further criteria for diagnosing OA include fibrosis, reduced range of joint motion, narrowing of joint space, and pain. Specific examples of osteoarthritis diagnostic and monitoring methods well known in the art include global assessment, Hip Disability and Osteoarthritis Outcome Score (HOOS), Knee Injury and Osteoarthritis Outcome Score (KOOS), and the Western Ontario McMaster University Osteoarthritis Index (WOMAC), as described, for example, in Nilsdotter et al. (BMC Musculoskeletal Disorders 4:10, 2003), Davis et al. (Osteoarthritis Cartilage 17(7):843-847, 2009, and Roos et al. (Health and Quality of Life Outcomes 1 :64, 2003); each of which is incorporated by reference herein.

Fibromyalgia

The invention features methods for treating a subject diagnosed with an inflammatory condition, such as fibromyalgia, with a tetracycline (e.g., doxycycline, minocycline, sarecycline, omadacycline, PTK- RA01 , PTK-MS01 , PTK-SMA2, another tetracycline described herein, or a derivative thereof; preferably doxycycline or sarecycline). Subjects may be diagnosed with fibromyalgia according to known and accepted guidelines, methods, or protocols, or by using the methods and kits described herein to detect the presence of fibromyalgia in the subject. The primary symptom of fibromyalgia is chronic, strongly systemic pain, or, even if partial, widespread chronic pain, the pain often being observed in muscular tissues and in the skin. According to the American College of Rheumatology, a patient satisfies diagnostic criteria for fibromyalgia if the following 3 conditions are met: (1 ) widespread pain index (WPI) >7 and symptom severity (SS) scale score >5 or WPI 3 - 6 and SS scale score >9; (2) symptoms have been present at a similar level for at least 3 months; and (3) the patient does not have a disorder that would otherwise explain the pain. Such systemic chronic pain is often not alone and may also be accompanied by a feeling of fatigue, malaise, depression, a feeling of anxiety, a feeling of morning stiffness, muscle stiffness, sleep disturbance or the like. In addition, symptoms such as headache, facial pain, cognitive impairment (lapse of memory, concentration deficit), gastrointestinal complaints (visceral pain, digestive system disturbance, flatulence), frequent urination, diarrhea, constipation, or

dysmenorrhea may also occur concomitantly. Constipation

The invention features methods for treating a subject diagnosed with an inflammatory condition, such as constipation, with a tetracycline (e.g., doxycycline, minocycline, sarecycline, omadacycline, PTK- RA01 , PTK-MS01 , PTK-SMA2, another tetracycline described herein, or a derivative thereof; preferably doxycycline or sarecycline). Subjects may be diagnosed with constipation according to known and accepted guidelines, methods, or protocols, or by using the methods and kits described herein to detect the presence of constipation in the subject. Constipation can be commonly diagnosed, for example, by the presence of symptoms in the subject, such as difficult and/or firm bowel movements, painful defecation, small and/or pellet-like stools, bloody stools, detection of scybala on abdominal palpation, bloating, distension, abdominal pain, impaction of fecal matter, bowel obstruction, headache, fatigue, or a sense of incomplete emptying. Diagnosis can by performed, e.g., by physical examination of the subject (e.g., rectal examination and colonoscopy) or by self-reporting of relevant symptoms by the subject.

Exemplary criteria for diagnosing constipation include the Rome II Criteria, which require at least two of the following symptoms for at least 12 weeks over the period of one year:

(i) straining with more than one-fourth of defecations,

(ii) hard stool with more than one-fourth of defecations,

(iii) feeling of incomplete evacuation with more than one-fourth of defecations,

(iv) sensation of anorectal obstruction with more than one-fourth of defecations,

(v) manual maneuvers to facilitate more than one-fourth of defecations,

(vi) fewer than three bowel movements per week, and/or

(vii) insufficient criteria for irritable bowel syndrome.

Adhesion

The invention features methods for treating a subject diagnosed with an inflammatory condition, such as an adhesion, with a tetracycline (e.g., doxycycline, minocycline, sarecycline, omadacycline, PTK- RA01 , PTK-MS01 , PTK-SMA2, another tetracycline described herein, or a derivative thereof; preferably doxycycline or sarecycline). Adhesions are fibrous bands that form between tissues and/or organs, and commonly occur as a result of, e.g., injury, surgery, and/or inflammation. The formation of an adhesion can occur when scar tissue extends from one tissue to another, e.g., across a space within the body, as a result, for example, of fibrin deposition in damaged tissues. Adhesions can form shortly after an injury or surgery (e.g., within a four hours after surgery), and can include attachment of internal organs or tissues to a surgical or injury site. An adhesion resulting from surgery can include, for example, post-operative adhesion after joint replacement surgery. Adhesions can occur in numerous tissues, including but not limited to a joint, tendon, ligament, eye, abdomen, pericardium, or pelvis. Joint adhesions can occur, for example, in a shoulder joint (e.g., adhesive capsulitis), knee joint, hip joint, ankle joint, elbow joint, or wrist joint.

Adhesion can be associated with a variety of signs and symptoms. For example, abdominal adhesion can be associated with, e.g., intestinal blockage, bloating, constipation, nausea, vomiting, abdominal pain, and/or cramps. Eye adhesion can be associated with glaucoma. A subject may be diagnosed with an adhesion according to known and accepted guidelines, methods, or protocols, or by using the methods and kits described herein to detect the presence of an adhesion in the subject. Non-bacterial inflammatory conditions

The invention features methods for treating a subject diagnosed with an inflammatory condition, such as a non-bacterial inflammatory condition (e.g., mTBI, OA, or fibromyalgia), with a tetracycline (e.g., doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK-SMA2, another tetracycline described herein, or a derivative thereof; preferably doxycycline or sarecycline). Subjects may be diagnosed with a non-bacterial inflammatory condition according to known and accepted guidelines, methods, or protocols, or by using the methods and kits described herein. Methods and kits of the present invention can be used to treat a subject for an inflammatory condition, including inflammatory conditions resulting from conditions other than bacterial infection. In certain embodiments, it may thus be desirable to determine whether an inflammatory condition results from a condition other than bacterial infection. Determining the presence of a bacterial infection can be done using standard diagnostic approaches well known in the art, including but not limited to PCR assays, enzyme-linked immunosorbent assay (ELISA), immunoassays, or cell culture. For example, immunoassays may be used to detect or monitor the expression of one or more polypeptides expressed by a pathogenic bacterium. Polyclonal or monoclonal antibodies capable of binding to such a polypeptide may be used in any standard immunoassay format (e.g., ELISA, Western blot, or RIA assay) to measure the level of the pathogenicity polypeptide.

Inflammatory biomarkers whose levels may be usefulness in the diagnosis and monitoring of such inflammatory conditions include, but are not limited to: TGF-βΙ , TGF^2, TGF^3, MMP1 , MMP3, MMP9, ILI A, IL1 B, TNFa, IL-6, interferon-gamma, IL-8, CX3CL1 , CXCL1 , CXCL2, CCL2-5, CCL21 , CXCL10, SDF-1 , IL-2, IL-17, IL-18, and IL-23 (preferably TGF-βΙ , TGF^2, or TGF^3; most preferably TGF-βΙ ). Expression levels of a TGF-β and one or more additional inflammatory biomarkers (e.g., an MMP, such as MMP1 or MMP3) can be, e.g., measured in a biological sample obtained from the same subject to, e.g., determine if the subject has an inflammatory condition or to monitor the progression of an inflammatory condition in the subject.

Determining inflammatory biomarker levels

The present invention features methods and kits involving determining the level of at least one inflammatory biomarker (e.g., an inflammatory cytokine), such as TGF-βΙ , TGF^2, TGF^3, MMP1 , MMP3, MMP9, IL1 A, IL1 B, TNFa, IL-6, interferon-gamma, IL-8, CX3CL1 , CXCL1 , CXCL2, CCL2-5, CCL21 , CXCL10, SDF-1 , IL-2, IL-17, IL-18, or IL-23, in a biological sample obtained from a subject. Preferably, the cytokine detected is TGF-βΙ , TGF^2, or TGF^3; most preferably TGF-βΙ . The biological sample may include, for example, blood, cerebrospinal fluid, or synovial fluid. Methods for obtaining such a biological sample from the subject are well known in the art. For example, inflammatory biomarkers (e.g., TGF-βΙ ) may be detected in blood samples, as described in, e.g., Anscher et al. (New England J. Med. 328(22): 1592-1598, 1993). Determining the levels of such biomarkers may be used to detect the presence of an inflammatory condition and/or a fibrotic condition in the subject. For example, TGF-β levels have been found to be elevated in synovial fluid obtained from OA patients, as described by, e.g., Remst et al. (Arthritis Rheum., E-pub ahead of print, 2013; incorporated herein in its entirety). Methods for determining the level of one or more inflammatory biomarkers (e.g., an inflammatory biomarker, such as TGF-βΙ ), such as those described herein, include, for example, determining the expression level of a polypeptide or nucleic acid corresponding to the biomarker of interest, measuring the activity of one or more proteins or nucleic acids (e.g., cytokines, downstream effectors of one or more cytokines, or enzymatic activity (for example, matrix metalloproteinase activity)), detecting a change in a biological activity, process, or structure affected by the biomarker (e.g., fibrosis in a tissue or organ affected by an inflammatory condition), or measuring the levels of a metabolite related to the biomarker or its biological activity in a biological sample. The level of the one or more inflammatory biomarkers can be compared to the level present in a reference sample, such as a biological sample obtained from a normal or healthy subject (e.g., a subject lacking an inflammatory condition), or to a normal level of the biomarker as shown in Table 1 . Analysis of biomarker level can take place prior to, during, or after a therapy. It is contemplated that biomarker levels can be used to titrate the therapeutic dose of a compound of the invention (e.g., doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK-SMA2, another tetracycline described herein, or a derivative thereof; preferably doxycycline or sarecycline) administered to a subject.

The level of one or more inflammatory biomarkers in a biological sample can be analyzed by a number of methodologies, many of which are known in the art and understood by the skilled artisan, including but not limited to, immunohistochemical and/or western blot analysis, immunoprecipitation, immunofluorescence, molecular binding assays, ELISA, ELI FA, fluorescence activated cell sorting (FACS), mass spectrometry, quantitative blood based assays (as for example serum ELISA) (to examine, for example, levels of protein expression), biochemical enzymatic activity assays, in situ hybridization, northern analysis and/or PCR analysis of mRNAs, as well as any one of the wide variety of assays that can be performed by gene and/or tissue array analysis or sequencing. Typical protocols for evaluating the status of genes and gene products (e.g., the genes and gene products corresponding to the inflammatory biomarkers listed in Table 1 ) are found, for example in Ausubel et al. eds., Current Protocols In Molecular Biology, 1995 (Units 2 [Northern Blotting], 4 [Southern Blotting], 15 [Immunoblotting], and 18 [PCR Analysis]). Multiplexed immunoassays such as those available from Rules Based Medicine or Meso Scale Discovery (MSD), Multiple Reaction Monitoring (MRM), multiplexed RTPCR, IHC or multiplex variation of any of the above-mentioned assays may also be used.

The expression of a protein of one or more genes in a sample can be, e.g., examined using immunohistochemistry ("IHC") and staining protocols. IHC staining of tissue sections has been shown to be a reliable method of assessing or detecting presence of proteins in a sample. IHC and IFC techniques use an antibody to probe and visualize cellular antigens in situ, generally by chromogenic or fluorescent methods. The tissue sample may be fixed (i.e., preserved) by conventional methodology (see, e.g., Luna et al. eds., "Manual of Histological Staining Method of the Armed Forces Institute of Pathology," 3rd edition, 1960, The Blakston Division McGraw-Hill Book Company, New York; Mikel et al., eds., "The Armed Forces Institute of Pathology Advanced Laboratory Methods in Histology and Pathology," 1994, Armed Forces Institute of Pathology, American Registry of Pathology, Washington, D.C.). One of skill in the art will appreciate that the choice of a fixative is determined by the purpose for which the sample is to be histologically stained or otherwise analyzed. By way of example, neutral buffered formalin, Bouin's or paraformaldehyde, may be used to fix a sample. Generally, the sample is first fixed and is then dehydrated through an ascending series of alcohols, infiltrated and embedded with paraffin or other sectioning media so that the tissue sample may be sectioned. Alternatively, one may section the tissue and fix the sections obtained. The primary and/or secondary antibody used for immunohistochemistry typically will be labeled with a detectable moiety, such as a radioisotope, a colloidal gold particle, a fluorescent label, a chromogenic label, or an enzyme-substrate label.

In alternative methods, the sample may be contacted with an antibody specific for the gene or biomarker under conditions sufficient for an antibody-biomarker complex to form, and then detecting the complex. The presence of the biomarker may be detected in a number of ways, such as by Western blotting and ELISA procedures for assaying a wide variety of tissues and samples, including plasma or serum. A wide range of immunoassay techniques using such an assay format are available (see, e.g., U.S. Pat. No. 4,016,043, U.S. Pat. No. 4,424,279, and U.S. Pat. No. 4,018,653, each of which is incorporated herein by reference). These include both single-site and two-site or "sandwich" assays of the noncompetitive types, as well as in the traditional competitive binding assays. These assays also include direct binding of a labeled antibody to a target biomarker.

Another method involves immobilizing the target biomarkers (e.g., on a solid support) and then exposing the immobilized target to specific antibody, which may or may not contain a label. Depending on the amount of target and the strength of the label's signal, a bound target may be detectable by direct labeling with the antibody. Alternatively, a second labeled antibody, specific to the first antibody is exposed to the target-first antibody complex to form a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by a label, e.g., an enzyme, a fluorescent label, a chromogenic label, a radionuclide containing molecule (i.e., a radioisotope), or a chemiluminescent molecule.

Variations on the forward assay include a simultaneous assay, in which both sample and labeled antibody are added simultaneously to the bound antibody. These techniques are well known to those skilled in the art, including any minor variations as will be readily apparent. In a typical forward sandwich assay, a first antibody having specificity for the biomarker is either covalently or passively bound to a solid surface (e.g., a glass or a polymer surface, such as those with solid supports in the form of tubes, beads, discs, or microplates), and a second antibody is linked to a label that is used to indicate the binding of the second antibody to the molecular marker.

Another methodology for determining expression level in a sample is in situ hybridization, for example, fluorescence in situ hybridization (FISH) (see, e.g., Angerer et al., Methods Enzymol. 152: 649- 661 , 1987). Generally, in situ hybridization includes the following steps: (1 ) fixation of a biological sample to be analyzed; (2) pre-hybridization treatment of the biological sample to increase accessibility of target DNA and to reduce non-specific binding; (3) hybridization of the mixture of nucleic acids to the nucleic acid in the biological sample; (4) post-hybridization washes to remove nucleic acid fragments not bound in the hybridization; and (5) detection of the hybridized nucleic acid fragments. The binding agents (e.g., probes) used in such applications are typically labeled, for example, with radioisotopes or fluorescent labels. Preferred probes are sufficiently long, for example, from about 50, 100, or 200 nucleotides to about 1000 or more nucleotides, to enable specific hybridization with the target nucleic acid(s) under stringent conditions. Another methodology for determining expression level in a sample is Immuno-PCR (IPCR). IPCR employs conjugates between nucleic acid marker sequences and antibodies together with PCR, which is widely applied for detecting various types of targets including proteins (see Sano et al., Science 258: 120- 122, 1992; U.S. Pat. No. 5,665,539; Niemeyer et al., Trends in Biotechnology 23: 208-216, 2005; U.S. Pat. Pub. No. 2005/0239108; and Ye et al., Journal of Environmental Science 22: 796-800, 2010).

Alternative methods for determining the expression level in a sample include bead based multiplex assays, such as Luminex, and multiple reaction monitoring (MRM) mass spectrometry based assays.

Amplification-based assays also can be used to measure the expression level of one or more genes. In such assays, the nucleic acid sequences of the gene act as a template in an amplification reaction (for example, a polymerase chain reaction (PCR) or quantitative PCR). In a quantitative amplification, the amount of amplification product will be proportional to the amount of template in the original sample. Comparison to appropriate controls provides a measure of the expression level of the gene, corresponding to the specific probe used, according to the principles discussed above. Methods of real-time quantitative PCR using TaqMan probes are well known in the art. Detailed protocols for realtime quantitative PCR are provided, for example, in Gibson et al., Genome Res. 6: 995-1001 , 1996, and in Heid et al., Genome Res. 6: 986-994, 1996.

A TaqMan-based assay also can be used to quantify expression level. TaqMan-based assays use a fluorogenic oligonucleotide probe that contains a 5' fluorescent dye and a 3' quenching agent. The probe hybridizes to a PCR product, but cannot itself be extended due to a blocking agent at the 3' end. When the PCR product is amplified in subsequent cycles, the 5' nuclease activity of the polymerase, for example, AmpliTaq, results in the cleavage of the TaqMan probe. This cleavage separates the 5' fluorescent dye and the 3' quenching agent, thereby resulting in an increase in fluorescence as a function of amplification.

Other suitable amplification methods include, but are not limited to, ligase chain reaction (LCR)

(see, e.g., Wu and Wallace, Genomics 4: 560-569, 1989; Landegren et al., Science 241 : 1077-1080, 1988; and Barringer et al., Gene 89: 1 17-122, 1990), transcription amplification (see, e.g., Kwoh et al., Proc. Natl. Acad. Sci. 86: 1 173-1 177, 1989), self-sustained sequence replication (see, e.g., Guatelli et al., Proc. Natl. Acad. Sci. 87: 1874-1878, 1990), dot PCR, and linker adapter PCR.

Expression levels may also be determined using microarray-based platforms (e.g., single- nucleotide polymorphism (SNP) arrays), as microarray technology offers high resolution. Details of various microarray methods can be found in the literature. See, for example, U.S. Pat. No. 6,232,068 and Pollack et al., Nat. Genet. 23: 41 -46, 1999.

Expression levels may also be determined using next generation sequencing platforms (e.g., RNA-Seq), as described in Mortazavi et al., Nat. Methods 5: 621 -628, 2008, hereby incorporated by reference. RNA-Seq is a robust technology for monitoring expression by direct sequencing the RNA molecules in a sample. Briefly, this methodology includes fragmentation of RNA to an average length of 200 nucleotides, conversion to cDNA by random priming, and synthesis of double-stranded cDNA (e.g., using the Just cDNA DoubleStranded cDNA Synthesis Kit from Agilent Technology). Then, the cDNA is converted into a molecular library for sequencing by addition of sequence adapters for each library (e.g., from lllumina®/Solexa), and the resulting 50-100 nucleotide reads are mapped onto the genome. Methods of the invention further include protocols which examine the presence and/or expression of mRNAs of one or more genes, in a tissue or cell sample. Methods for the evaluation of mRNAs in cells are well known and include, for example, hybridization assays using complementary DNA probes (such as in situ hybridization using labeled riboprobes specific for the one or more genes, northern blot and related techniques) and various nucleic acid amplification assays (such as RT-PCR using complementary primers specific for one or more of the genes, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA, and the like). The probes for these assays may be labeled for detection according to methods known in the art.

Tissue or cell samples from mammals can be conveniently assayed for mRNAs using Northern, dot blot or PCR analysis. For example, RT-PCR assays such as quantitative PCR assays are well known in the art. In an illustrative embodiment of the invention, a method for detecting a target mRNA in a biological sample comprises producing cDNA from the sample by reverse transcription using at least one primer; amplifying the cDNA so produced using a target polynucleotide as sense and antisense primers to amplify target cDNAs therein; and detecting the presence of the amplified target cDNA using

polynucleotide probes. In some embodiments, primers and probes comprising the sequences described herein are used to detect expression of one or more genes, as described herein. In addition, such methods can include one or more steps that allow one to determine the levels of target mRNA in a biological sample (e.g., by simultaneously examining the levels a comparative control mRNA sequence of a "housekeeping" gene such as an actin family member or any control gene described herein, such as GAPDH). Optionally, the sequence of the amplified target cDNA can be determined. The primers for these assays may be labeled for detection according to methods known in the art.

Optional methods of the invention include protocols which examine or detect mRNAs, such as target mRNAs, in a tissue or cell sample by microarray technologies. Using nucleic acid microarrays, test and control mRNA samples from test and control tissue samples are reverse transcribed and labeled to generate cDNA probes. The probes can then hybridized to an array of nucleic acids immobilized on a solid support. The array can be configured such that the sequence and position of each member of the array is known. For example, a selection of genes whose expression correlate with the presence of PDAC, an increased likelihood of developing PDAC, or increased severity of PDAC can be arrayed on a solid support. Hybridization of a labeled probe with a particular array member indicates that the sample from which the probe was derived expresses that gene. Differential gene expression analysis of disease tissue can provide valuable information. Microarray technology utilizes nucleic acid hybridization techniques and computing technology to evaluate the mRNA expression profile of thousands of genes within a single experiment (see, e.g., WO 01/75166 published October 1 1 , 2001 ; U.S. Pat. No. 5,700,637; U.S. Pat. No. 5,445,934; U.S. Pat. No. 5,807,522; Lockart, Nat. Biotechnol. 14: 1675-1680, 1996; Cheung et al., Nat. Genet. 21 (Suppl): 15-19, 1999). Thousands of genes are usually represented in a single array. A typical microarray experiment involves the following steps: 1 ) preparation of fluorescently labeled target from RNA isolated from the sample, 2) hybridization of the labeled target to the microarray, 3) washing, staining, and scanning of the array, 4) analysis of the scanned image and 5) generation of gene expression profiles. In forming an array, oligonucleotides can be either prefabricated and spotted to the surface or directly synthesized on to the surface (in situ). Commercially available microarray systems can be used, such as the Affymetrix GeneChip® system. Expression of a selected gene or biomarker in a tissue or cell sample and/or the activity of a gene or gene product of interest (e.g., an inflammatory biomarker) may be examined by way of functional or activity-based assays. For instance, if the biomarker is an enzyme, one may conduct assays known in the art to determine or detect the presence of the given enzymatic activity in the tissue or cell sample. A non-limiting example of a biological activity that may be measured to determine TGF-β levels is the growth of mink-lung epithelial cells in vitro (Anscher et al., New England J. Med. 328(22): 1592-1598, 1993).

Any of the methods herein can be adapted to include a solid support. Exemplary solid supports include a glass or a polymer surface, including one or more of a well, a plate, a wellplate, a tube, an array, a bead, a disc, a microarray, or a microplate. In particular, the solid supported can be adapted to allow for automation of any one of the methods described herein (e.g., PCR). Alternatively microfluidics or microdroplets could be used.

Detection of amplification, overexpression, or overproduction of, for example, a gene or gene product can also be used to provide prognostic information or guide therapeutic treatment. Such prognostic or predictive assays can be used to determine prophylactic treatment of a subject prior to the onset of symptoms or stratification of patients to particular treatment protocols.

The diagnostic methods described herein can be used individually or in combination with any other diagnostic method described herein for a more accurate diagnosis of the presence or severity of a condition (e.g., an inflammatory condition, such as mTBI, OA, or fibromyalgia).

Methods of Treatment

Inflammation is associated with a wide range of medical conditions. The present invention features methods for treating a subject (e.g., a human) for an inflammatory condition, preferably an inflammatory condition resulting from a condition other than bacterial infection, such as mTBI, OA, fibromyalgia, diabetic adhesive capsulitis, hypermobility syndrome, exercise intolerance, arthropathy, inflammatory bowel disease, constipation, diabetes-associated inflammation, adhesion (e.g., an adhesion as described herein, such as post-operative adhesion, post-operative adhesion after joint replacement surgery, and abdominal adhesion), Ehlers-Danlos Syndrome, sarcopenia, sarcoidosis, and liver fibrosis. Preferably the inflammatory condition is mTBI, OA, fibromyalgia, constipation, or adhesion. The subject may have, for example, an elevated level of at least one TGF-β (e.g., TGF-βΙ , TGF^2, or TGF^3) relative to a level in a normal subject (e.g., a subject previously determined to have the elevated level of the at least one TGF-β). The inflammatory condition may result, for example, from a condition other than an autoimmune disorder. Such inflammatory conditions include those associated with observable fibrosis or fibrosis detectable by an increase in one or more inflammatory biomarkers (e.g., an inflammatory cytokine, such as TGF-βΙ ). The inflammatory condition may also involve, e.g., an extracellular matrix (ECM) associated crosslinking condition (e.g., diabetes-associated inflammation), such as aberrant ECM production and/or maintenance by fibroblasts during repair of damaged tissue.

The methods of treatment disclosed herein are based, inter alia, on the inventor's discovery that tetracyclines (e.g., doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK- SMA2, another tetracycline described herein, or a derivative thereof; preferably doxycycline or sarecycline) may be used to treat inflammatory conditions, in particular inflammatory conditions that promote fibrosis. The presence of an inflammatory condition in a subject may be determined by using diagnostic methods well known in the art, or by using methods for diagnosing inflammatory conditions described herein. The subjects may be treated with an effective amount of a tetracycline. The tetracycline can be, e.g., administered at a submicrobial dose. The amount of a tetracycline administered to a subject may be changed according to the severity of inflammation (e.g., as determined by measuring the expression level of one or more inflammatory biomarkers, e.g., a TGF-β, in the subject, according to the levels of inflammation shown in Table 1 ). For example, the amount of the tetracycline administered to the subject may be increased in the case of high inflammation, or if the inflammation increases in severity. Alternatively, the amount of the tetracycline may be, e.g., decreased in the case of low inflammation, or if the inflammation decreases in severity. The efficacy of the treatment may be monitored, e.g., by determining a change (e.g., a decrease) in the level of one or more inflammatory biomarkers (e.g., TGF-βΙ ) in the subject during and/or following therapy or by observing a decrease in fibrosis in the subject during and/or following therapy. Routes of administration

The invention features methods for treating subjects for inflammatory disorders by administering tetracycline compounds (e.g., doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK- MS01 , PTK-SMA2, another tetracycline described herein, or a derivative thereof; preferably doxycycline or sarecycline) to the subjects.

Routes of administration for the tetracycline compounds and pharmaceutical compositions comprising such compounds include, but are not limited to, oral, topical, transdermal, nasal, and systemic administration (such as, but not limited to, intravenous, intramuscular, subcutaneous, inhalation, rectal, buccal, vaginal, intraperitoneal, intraarticular, ophthalmic, otic, parenteral, or oral administration). In certain embodiments a tetracycline may be administered systemically (e.g., orally, or as an injectable) in accordance with standard methods known in the art.

Transdermal administration

A tetracycline (e.g., doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK-SMA2, another tetracycline described herein, or a derivative thereof; preferably doxycycline or sarecycline) may be delivered, e.g., through the skin using, for example, a transdermal drug delivery system. The transdermal drug delivery system may be, e.g., a transdermal "patch," in which the tetracycline is contained within a laminated structure that serves as a drug delivery device to be affixed to the skin. In such a structure, the drug composition may be contained in a layer, or reservoir, underlying an upper backing layer. The reservoir of a transdermal patch includes a quantity of an agent (e.g., a tetracycline) that is ultimately available for delivery to the surface of the skin. Thus, the reservoir may include an agent of the present invention (e.g., a tetracycline) in an adhesive on a backing layer of the patch, or in any of a variety of different matrix formulations known in the art. The patch may contain a single reservoir or multiple reservoirs.

A reservoir in a transdermal patch may comprise a polymeric matrix of a pharmaceutically acceptable contact adhesive material that serves to affix the system to the skin during drug delivery. Examples of suitable skin contact adhesive materials include, but are not limited to, polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates, and polyurethanes. Alternatively, the agent-containing reservoir and skin contact adhesive are present as separate and distinct layers, with the adhesive underlying the reservoir which, in this case, may be either a polymeric matrix as described above, a liquid or hydrogel reservoir, or another form of reservoir known in the art. The backing layer in these laminates, which serves as the upper surface of the device, preferably functions as a primary structural element of the patch and provides the device with a substantial portion of flexibility. The material selected for the backing layer is preferably substantially impermeable to the agent of the invention (e.g., a tetracycline) and to any other materials that are present. Preferably, the tetracycline (e.g., doxycycline, minocycline, sarecycline, or omadacycline; preferably doxycycline or sarecycline) is administered transdermal^ at a submicrobial dose.

Topical administration

A tetracycline (e.g., doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK-SMA2, another tetracycline described herein, or a derivative thereof; preferably doxycycline or sarecycline) can be administered topically. Formulations for topical delivery of tetracyclines include, but are not limited to, ointments, gels, sprays, fluids, and creams. Ointments are semisolid preparations that are typically based on petrolatum or other petroleum derivatives. Creams including an agent of the invention, e.g., a tetracycline, are typically viscous liquids or semisolid emulsions, e.g. oil-in-water or water-in-oil emulsions. Cream bases are typically water-washable and include an oil phase, an emulsifier, and an aqueous phase. The oil phase, also sometimes called the "internal" phase, of a cream base is generally comprised of petrolatum and a fatty alcohol, e.g. cetyl alcohol or stearyl alcohol; the aqueous phase usually, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant. The emulsifier in a cream formulation is generally a nonionic, anionic, cationic, or amphoteric surfactant. The specific ointment or cream base to be used may be selected to provide for optimum drug delivery according to the art. As with other carriers or vehicles, an ointment base may be inert, stable, non-irritating, and non-sensitizing. Preferably, the tetracycline (e.g., doxycycline, minocycline, sarecycline, or omadacycline; preferably doxycycline or sarecycline) is administered topically at a submicrobial dose. CNS administration

It may be desirable to deliver a tetracycline to the central nervous system and/or the brain. In embodiments including systemic administration, this could require that the agent cross the blood brain barrier. In various embodiments this may be facilitated by co-administering the tetracycline (e.g., doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK-SMA2, another tetracycline described herein, or a derivative thereof; preferably doxycycline or sarecycline) with carrier molecules such as cationic dendrimers or arginine-rich peptides, which may carry the agent over the blood brain barrier.

A tetracycline may be delivered directly to the brain by, e.g., administration through the implantation of a biocompatible release system (e.g., a reservoir), by direct administration through an implanted cannula, by administration through an implanted or partially implanted drug pump, or mechanisms of similar function known the art. In certain embodiments, the tetracycline may be systemically administered (e.g., injected into a vein). In other embodiments, it is expected that the tetracycline will be transported across the blood brain barrier without the use of additional compounds included in a pharmaceutical composition to enhance transport across the blood brain barrier. Preferably, the tetracycline (e.g., doxycycline, minocycline, sarecycline, or omadacycline; preferably doxycycline or sarecycline) is administered to the CNS at a submicrobial dose.

Oral and rectal administration

A tetracycline of the invention, such as doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK-SMA2, another tetracycline described herein, or a derivative thereof (preferably doxycycline or sarecycline), may be administered orally. Tetracyclines may be formulated for oral administration in tablets, capsules, elixirs or syrups. Tetracyclines may be formulated for rectal administration in the form of suppositories. In cases where the compound in itself is not sufficiently soluble to be dissolved, a solubilizer such as ethanol can be applied. Other suitable formulations and modes of administration are known or may be derived from the art. Preferably, the tetracycline (e.g., doxycycline, minocycline, sarecycline, or omadacycline; preferably doxycycline or sarecycline) is administered orally or rectally at a submicrobial dose.

Formulation

Compounds of the invention (e.g., tetracyclines, such as doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK-SMA2, another tetracycline described herein, or a derivative thereof; preferably doxycycline or sarecycline) for use in treatment of human or animal subjects for inflammatory conditions can be formulated as pharmaceutical or veterinary compositions, respectively. Depending on the subject to be treated, the mode of administration, and the type of treatment desired (e.g., prevention, prophylaxis, or therapy) the tetracycline may be formulated in ways consonant with these parameters. A summary of such techniques is found in Remington: The Science and Practice of Pharmacy, 21^st Edition, Lippincott Williams & Wilkins, (2005); and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York, each of which is incorporated herein by reference.

Tetracyclines described herein may be present in amounts totaling 1 -95% by weight of the total weight of the composition. The composition may be provided in a dosage form that is suitable for intraarticular, oral, parenteral (e.g., intravenous, intramuscular), rectal, cutaneous, subcutaneous, topical, transdermal, sublingual, nasal, vaginal, intravesicular, intraurethral, intrathecal, epidural, aural, or ocular administration, or by injection, inhalation, or direct contact with the nasal, genitourinary, gastrointesitnal, reproductive or oral mucosa. Thus, the pharmaceutical composition may be in the form of, e.g., tablets, capsules, pills, powders, granulates, suspensions, emulsions, solutions, gels including hydrogels, pastes, ointments, creams, plasters, drenches, osmotic delivery devices, suppositories, enemas, injectables, implants, sprays, preparations suitable for iontophoretic delivery, or aerosols. The compositions may be formulated according to conventional pharmaceutical practice.

Tetracyclines of the invention may be prepared and used as pharmaceutical compositions comprising an effective amount of a compound described herein and a pharmaceutically acceptable carrier or excipient, as is well known in the art. In some embodiments, the composition includes at least two different pharmaceutically acceptable excipients or carriers.

Formulations may be prepared in a manner suitable for systemic administration or topical or local administration. Systemic formulations include those designed for injection (e.g., intramuscular, intravenous or subcutaneous injection) or may be prepared for transdermal, transmucosal, or oral administration. The formulation will generally include a diluent as well as, in some cases, adjuvants, buffers, preservatives and the like. The compounds can be administered also in liposomal compositions or as microemulsions.

For injection, formulations can be prepared in conventional forms as liquid solutions or suspensions or as solid forms suitable for solution or suspension in liquid prior to injection or as emulsions. Suitable excipients include, for example, water, saline, dextrose, glycerol and the like. Such compositions may also contain amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, such as, for example, sodium acetate, sorbitan monolaurate, and so forth.

Various sustained release systems for drugs have also been devised. See, for example, U.S.

Pat. No. 5,624,677, which is herein incorporated by reference.

Systemic administration may also include relatively noninvasive methods such as the use of suppositories, transdermal patches, transmucosal delivery and intranasal administration. Oral administration is also suitable for compounds of the invention. Suitable forms include syrups, capsules, and tablets, as is understood in the art.

Each compound of a combination therapy, as described herein, may be formulated in a variety of ways that are known in the art. For example, the first and second agents of the combination therapy may be formulated together or separately. For example, the first agent may be a tetracycline, while the second agent may be an anti-inflammatory agent (e.g., an NSAID).

The individually or separately formulated agents can be packaged together as a kit. Non-limiting examples include, but are not limited to, kits that contain, e.g., two pills, a pill and a powder, a suppository and a liquid in a vial, two topical creams, etc. The kit can include optional components that aid in the administration of the unit dose to patients, such as vials for reconstituting powder forms, syringes for injection, customized IV delivery systems, inhalers, etc. Additionally, the unit dose kit can contain instructions for preparation and administration of the compositions. The kit may be manufactured as a single use unit dose for one patient, multiple uses for a particular patient (at a constant dose or in which the individual compounds may vary in potency as therapy progresses); or the kit may contain multiple doses suitable for administration to multiple patients ("bulk packaging"). The kit components may be assembled in cartons, blister packs, bottles, tubes, and the like.

Formulations for oral use include tablets containing the active ingredient(s) in a mixture with nontoxic pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates, or alginic acid); binding agents (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, carboxymethylcellulose sodium, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents, glidants, and antiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc). Other pharmaceutically acceptable excipients can be colorants, flavoring agents, plasticizers, humectants, buffering agents, and the like.

Two or more compounds may be mixed together in a tablet, capsule, or other vehicle, or may be partitioned (e.g., a tetracycline, such as doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK-SMA2, another tetracycline described herein, or a derivative thereof; preferably doxycycline or sarecycline; in which the tetracycline is administered in combination with a second agent, such as an anti-inflammatory agent, e.g., an NSAID). In one example, the first compound is contained on the inside of the tablet, and the second compound is on the outside, such that a substantial portion of the second compound is released prior to the release of the first compound.

Formulations for oral use may also be provided as chewable tablets, or as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose,

microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin), or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil. Powders, granulates, and pellets may be prepared using the ingredients mentioned above under tablets and capsules in a conventional manner using, e.g., a mixer, a fluid bed apparatus or a spray drying equipment.

Dissolution or diffusion controlled release can be achieved by appropriate coating of a tablet, capsule, pellet, or granulate formulation of compounds (e.g., a tetracycline, such as doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK-SMA2, another tetracycline described herein, or a derivative thereof; preferably doxycycline or sarecycline), or by incorporating the compound into an appropriate matrix. A controlled release coating may include one or more of the coating substances mentioned above and/or, e.g., shellac, beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl distearate, glycerol palmitostearate, ethylcellulose, acrylic resins, dl-polylactic acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone, polyethylene, polymethacrylate, methylmethacrylate, 2-hydroxymethacrylate, methacrylate hydrogels, 1 ,3 butylene glycol, ethylene glycol methacrylate, and/or polyethylene glycols. In a controlled release matrix formulation of a compound of the invention (e.g., a tetracycline, such as doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK-SMA2, another tetracycline described herein, or a derivative thereof; preferably doxycycline or sarecycline), the matrix material may also include, e.g., hydrated methylcellulose, carnauba wax and stearyl alcohol, carbopol 934, silicone, glyceryl tristearate, methyl acrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and/or halogenated fluorocarbon.

The liquid forms in which the compounds and compositions of the present invention (e.g., a tetracycline, such as doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK- SMA2, another tetracycline described herein, or a derivative thereof; preferably doxycycline or sarecycline) can be incorporated for administration orally include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles. Generally, when administered to a human, the dosage of any of the compounds of the invention (e.g., tetracyclines, such as doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK-SMA2, another tetracycline described herein, or a derivative thereof; preferably doxycycline or sarecycline) will depend on, e.g., the nature of the compound and the route of administration, which can readily be determined by one skilled in the art. Tetracyclines for treatment of inflammatory disorders according to the methods and kits of the invention will generally be administered at submicrobial doses. Typically, such dosage is normally about 0.001 mg to 2000 mg per day, desirably about 1 mg to 200 mg per day, and more desirably about 10 mg to 100 mg per day. The timing of administration of the tetracycline may include, for example, administration once-monthly, once-weekly, once-daily, twice-daily, once every 48 hours, once every 36 hours, once every 24 hours, once every 12 hours, once every 6 hours, once every 4 hours, once every 3 hours, once every 2 hours, or once every hour.

Combination Therapy

Compounds of the invention (e.g., a tetracycline, such as doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK-SMA2, another tetracycline described herein, or a derivative thereof; preferably doxycycline or sarecycline) may be formulated and employed in combination therapies for the treatment of an inflammatory condition, such as mTBI, OA, or fibromyalgia. In such combination therapies, therapeutic agents of the invention may be formulated with or administered concurrently with, prior to, or subsequent to, one or more other desired therapies (e.g., therapeutic agents or medical procedures). The particular combination of therapies to employ in a combination regimen will take into account compatibility of the desired therapeutics and/or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed may achieve a desired effect for the same disorder, or they may achieve different effects (e.g., control of any adverse effects).

In general, for use in treatment, the tetracyclines described herein may be used alone, as mixtures of two or more compounds, or in combination with other agents, compounds, and/or pharmaceuticals. Examples of other agents that can be combined with the compounds described herein include agents that are known to be used for the treatment of an inflammatory condition. Another example of a potential agent to combine with the compounds described herein would include agents for the treatment of different yet associated or related symptoms or indications. Examples of agents that can be used in a combination therapy with a tetracycline of the invention (e.g., doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK-SMA2, another tetracycline described herein, or a derivative thereof; preferably doxycycline or sarecycline) include, but are not limited to, non-steroidal anti-inflammatory drug (NSAID), a COX-2 inhibitor, capsaicin, and gabapentin. Depending on the mode of administration, the agents will be formulated into suitable compositions to permit facile delivery. Each component of a combination therapy may be formulated in a variety of ways that are known in the art. For example, the first and second agents of the combination therapy may be formulated together or separately. Desirably, the first and second agents are formulated together for the simultaneous or near simultaneous administration of the agents.

The combination therapy may provide "synergy" and prove "synergistic," i.e., the effect achieved when the active ingredients used together is greater than the sum of the effects that results from using the compounds separately. A synergistic effect may be attained when the active ingredients are: (1 ) co- formulated and administered or delivered simultaneously in a combined, unit dosage formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by some other regimen. When delivered in alternation therapy, a synergistic effect may be attained when the compounds, agents, and/or treatments are administered or delivered sequentially, e.g., by different injections in separate syringes. In general, during alternation therapy, an effective dosage of each active ingredient is administered sequentially, i.e., serially, whereas in combination therapy, effective dosages of two or more active ingredients are administered together. Suitable dosages for any of the above co-administered agents are those presently used and may be lowered due to the combined action (synergy) of a compound of the present invention and other co-administered agents or treatments.

The compound of the present invention and additional agent are suitably administered to the patient at one time or over a series of treatments.

Kits of the invention

The present invention relates to a kit for conveniently and effectively diagnosing and/or treating a subject for an inflammatory disorder, such as mTBI, OA, or fibromyalgia. In general, the kit may include one or more containers filled with one or more of the compounds of the invention (e.g., tetracyclines, such as doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK-SMA2, another tetracycline described herein, or a derivative thereof; preferably doxycycline or sarecycline). The tetracycline may be formulated as described in this application. The tetracycline may be prepared for routes of administration as described herein. Such kits may be preferably suited for the delivery of oral forms, such as tablets or capsules, transdermal forms, such as patches, or topical forms, such as ointments, gels, sprays, fluids, and creams. Such a kit preferably includes a number of unit dosages, and may also include a card having the dosages oriented in the order of their intended use. A memory aid can be provided, for example, in the form of numbers, letters, or other markings or with a calendar insert, designating the days in the treatment schedule in which the dosages can be administered. Alternatively, placebo dosages, or calcium dietary supplements, either in a form similar to or distinct from the dosages of the pharmaceutical compositions, can be included to provide a kit in which a dosage is taken every day. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

The kit may further include a device for detecting a level of at least on inflammatory biomarker (e.g., an inflammatory cytokine), such as those listed in Table 1 (e.g., TGF-βΙ , TGF^2, or TGF^3; preferably TGF-βΙ ), in a biological sample. Such kits for detecting biomarker levels include those well known in the art. Examples include the TGF beta 1 Human ELISA Kit (Abeam, Cat. No. ab100647), the Human TGF-beta 1 Quantikine ELISA Kit (R&D Systems, Cat. No. DB100B), or the TGF-βΙ Multispecies ELISA Kit (Life Technologies, Cat. No. KAC1688).

Examples

The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed modified to yield essentially the same or similar results. Example 1. Treatment of mTBI with doxycycline

We have completed a case series which shows dramatic improvement of post-mTBI symptoms after once-daily doxycycline treatment. The patients were treated with doxycycline for three weeks with headache post-concussion as their main complaint. One patient, a college student, reported having had headaches for three years following a concussion. The second patient, a school teacher, had 4 months of headaches. The third patient, a college hockey player, presented with headaches, sleep disturbance, difficulty with concentration, and was unable to attend class as a result. The hockey player had been previously treated with Adderall and diazepam (Valium), without success. Each patient was treated with 100 mg of doxycycline, once per day, for a period of one month (30 days). All three patients showed significant improvement in symptoms after treatment with doxycycline. The college student showed an 80% reduction in headaches after one month of doxycycline treatment. The school teacher showed initial improvement, but stopped the medication. She recently returned to the clinic with increasing headaches and has since been placed back on the doxycycline regimen. The hockey player showed complete resolution of all symptoms after two weeks of doxycycline treatment.

Example 2. Role of TGF-β in mTBI

We hypothesize that, due to immunoexcitotoxicity, peripheral circulating levels of TGF-β will be elevated. There are very few pharmacologic treatment options for mTBI. NSAIDs and other anti- inflammatory agents have not been largely successful. We have hypothesized that mitigating the secretion of TGF-β may be a beneficial pharmacological approach in prevention of development of post- mTBI neurologic complications.

A randomized, placebo-controlled study will be conducted to investigate the effect of doxycycline on symptoms of patients with mTBI. We plan to recruit 100 patients for the study and similar number of matched control subjects. Both male and female patients will be enrolled for the study. We have a large patient population base of mTBI visiting the clinic, mainly related to the sports orientation of the city of Boston. The study populations are patients with confirmed diagnosis of concussion/ mTBI according to IMPACT criteria. Blood samples will be drawn and biobanked. We have already submitted a first batch of blood samples for TGF-β ELISA at the local laboratory facility at Brigham and Women's Hospital. The output parameters evaluated will include peripheral circulating levels of pro-inflammatory cytokines including TGF-β and SMAD (phosphorylated Smad2/3 and Smad4) using highly sensitive ELISA.

Genomic DNA will be obtained from peripheral circulating white cells and assayed by polymerase chain reaction for polymorphism of TGF-β genes (CC genotype) at the Center for Human Genetics, Inc (Boston). Correlation analyses will be performed between genomic variations of TGF-β gene expression, patient symptoms and peripheral circulating levels of TGF-β before and after treatment with

subantimicrobial doses of doxycycline. This will provide additional evidence whether certain genotypes predispose one to develop post-mTBI symptoms of neurocognitive dysfunction. This will form the first baseline study to test whether TGF-β represents a biomarker for prognosticating development of post- mTBI symptoms, which we will thereafter examine in a military based population. Example 3. Diagnosis of mTBI based on blood level of TGF-βΙ

A patient that presents with mTBI or symptoms of mTBI may be assessed for levels of an inflammatory biomarker (e.g., an inflammatory cytokine, such as those shown in Table 1 ). For example, a blood sample may be drawn from the patient and examined for serum levels of TGF-βΙ . If the level of TGF-βΙ in the blood sample is found to be elevated relative to a standard, such as the normal level of TGF-βΙ shown in Table 1 (0-20 ng/ml), or to the level of TGF-βΙ found in a blood sample from a healthy subject, then we would conclude that the patient is likely to be responsive to treatment with doxycycline. A patient showing such elevated levels of TGF-βΙ would thus be treated with doxycycline and then monitored for improvement in mTBI symptoms and for normalization of serum TGF-βΙ levels.

Example 4. Diagnosis of mTBI using imaging techniques

Diffusion tensor imaging (DTI) is a new neuroimaging technique that is sensitive to subtle changes in white matter fiber tracts and is capable of revealing microstructural axonal injuries (Fox et al., Neurol. Res., 35(3): 223-232, 2013; Shenton et al., Brain Imaging Behav., 6(2): 137-192, 2012; Niogi and Mukherjee, J. Head Trauma Rehab., 25(4): 241 -255, 2010), which are also potentially responsible for persistent postconcussive symptoms. Diffusion Weighted Imaging (DWI)/ Diffusion Tensor Imaging (DTI) uses a special type of MRI sequence that utilizes the diffusion properties of water to detect

microstructural tissue architecture. It is the best imaging technique available for detecting white matter integrity/damage, able to detect microscopic white matter damage and trace specific tracts of the brain (e.g., corpus callosum, superior longitudinal fasciculus, uncinate). Quantification of pathology using DTI is based on measures that calculate the amount of restriction of water movement in the brain, which is determined to a large extent by the tissue being measured. For example, the movement of water is unrestricted in a medium such as CSF, where it diffuses equally in all directions (i.e., isotropic). However, in white matter, the movement of water is more restricted by axonal membranes, myelin sheaths, microtubules, neurofilaments, etc. In white matter, this restriction is dependent on the directionality of the axons (i.e., diffusion is not equal in all directions) and is referred to as anisotropic diffusion. Using tensors, adapted from the field of engineering, the average shape of the diffusion is characterized as more or less spherical when there is no impediment to water diffusion, as for example in CSF (i.e., unrestricted water is free to diffuse in all directions: isotropic). However, the average shape of the diffusion becomes more elongated, or cigar shaped, when there is a preferred orientation in which water is restricted, as for example in white matter. Here, water diffuses freely in directions parallel to axons but it is restricted in directions that are perpendicular to the axons, which results in the magnitude of the diffusion along the axons being larger than the two perpendicular directions, leading to an elongated ellipsoidal shape of the diffusion tensor, described as anisotropic. The measurement of the distance that water diffuses, over a given period of time, for at least six non-collinear directions, makes it possible to reconstruct a diffusion tensor (and the associated ellipsoid) that best describes water diffusion within a given voxel. Consequently, the volume (size) and shape of the ellipsoid can be calculated, and this provides important information about the diffusion properties, and hence about microstructural aspects of brain tissue. There are various ways that the shape and size of a diffusion ellipsoid can be quantified, but the two most common indices used are Fractional Anisotropy (FA) for shape, and Mean Diffusivity (MD) for size. FA is a scalar measure that ranges from 0 to 1 , with 0 being completely isotropic, meaning that water diffuses equally in all directions, and 1 depicting the most extreme anisotropic scenario in which molecules are diffusing along a single axis. Accordingly, in CSF and gray matter, as noted above, the direction of water is equal in all directions (i.e., isotropic), and the value is close to 0. In contrast, in white matter, for example in the corpus callosum, the water is relatively free along the axons, but restricted perpendicular to the axons, and therefore more anisotropic, with FA being closer to 1 . Thus in white matter, reduced FA is generally thought to reflect loss of white matter integrity that may reflect damage to myelin or axon membrane damage, or perhaps reduced axonal packing density, and/or reduced axonal coherence. Because of our ability to recruit large number of patients with mild TBI and ability to follow them, we would be able to obtain these images, analyze them critically and create a registry.

We hypothesize that immunoexcitotoxicity is a significant pathophysiologic mechanism for development of neurocognitive decline and neurologic complications following mTBI (Blaylock and Maroon, Surg. Neurol. Int., 2: 107, 201 1 ). This may be reflected in the peripheral blood by increasing circulating levels of TGF-β. TGF-β levels will also be correlated with any subtle changes seen during brain imaging during routine follow-up visits. Our long-term collaboration with Shields Inc provides us the ability for optimal cost imaging. Serial correlative DTI will be performed and analyzed for any possible changes and correlate with peripheral blood biomarkers. These experiments, taken together, explore a rational pharmacotherapy using doxycycline, which via its mechanism of TGF-β inhibition can impede the progress of neurocognitive decline in post-mTBI patients. Example 5. Treatment of OA with doxycycline

Post operative adhesive capsulitis in the knee is a common poor outcome of surgery. Often, the patient has had no problem with the contralateral knee replacement. The occurrence of an inflammatory capsulitis is unpredictable but leads to significant disability in patients. Patient "DC" was a 72 year old male that presented 3 weeks post total knee replacement. DCs knee was almost immobile and CAT scan of the knee revealed joint effusion, joint capsule swelling and normal prosthesis. Infection could not be ruled out but there was no evidence of bony abnormality. The patient was placed on a submicrobial dose of doxycycline (100 mg per day) and 3 weeks later his motion had improved from a total ROM of 85 degrees to 1 10 degrees, with minimal swelling. We theorize that loss of muscle balance, aponeurosis, and sarcopenia lead to osteoarthritis and loss of cartilage protection. We further hypothesize that, in addition to imbalance of muscle function, structural muscle changes might interfere with the

chondroprotective effect of the musculature and thus contribute to the development of OA. Here, we show that tetracyclines, such as doxycycline, can be used to ameliorate the fibrotic state of the muscle architecture in OA patients.

This study hopes to expose a new paradigm for not only treatment of OA but a new model of disease progression. The rationale of this study focuses on the hypothesis that postinjury fibrotic features in OA result from over expression of cytokines like TGF-β, and that peripherally administered doxycycline can inhibit synthesis of TGF-β with subsequent improvement in muscle integrity with improvement in joint loading. Thus, it is believed that TGF-β can be used as a peripheral blood biomarker of muscle fibrosis in OA. Patients can be treated with a submicrobial dose of doxycycline as a therapeutic avenue for treatment of OA. The levels of TGF-β can also be assessed during therapy as a measure of therapeutic effectiveness. We hypothesize that the beneficial effects of doxycycline is related to muscle function improvement, in addition to any other additional effects it may have on joint cavity and its fibrous tissue contents.

Example 6. Additional study using tetracyclines to treat OA

We propose to perform a randomized, placebo-controlled study to investigate the effect of doxycycline on symptoms of patients with advanced knee osteoarthritis. We plan to recruit 60 male and female patients for the study and similar number of age and sex matched control subjects. Both male and female patients will be enrolled for the study. We have a large patient population base of advanced OA patients of all age groups visiting the clinic. Blood samples will be drawn and biobanked. The output parameters evaluated will include peripheral circulating levels of the pro-inflammatory biomarkers (e.g., pro-inflammatory cytokines), such as TGF-β and SMAD (phosphorylated Smad2/3 and Smad4) using highly sensitive ELISA. ELISA will be performed at the Biochemistry Core of Longwood Medical Area/Harvard Medical School. Genomic DNA will be obtained from peripheral circulating white cells and assayed by polymerase chain reaction for polymorphism of TGF-β genes (CC genotype) at the Center for Human Genetics, Inc (Boston, in collaboration with Prof. Aubrey Milunsky). Correlation analyses will be performed between genomic variations of TGF-β gene expression, patient symptoms, quantitation of muscle fibrosis and peripheral circulating levels of TGF-β before and after treatment with sub- antimicrobial doses of doxycycline. This will provide additional evidence whether certain genotypes predispose one to develop muscle fibrosis. This will form the first baseline study to test whether TGF-β represents a biomarker for prognosticating development of muscle fibrosis as an ongoing contributing pathophysiology to knee joint OA.

Ultrasonic imaging will allow us to obtain dynamic imaging of skeletal muscles in healthy subjects as well as patients with OA. Numerous joint maneuvers and muscle lengthening will be performed, and dynamic USG of the muscles will be obtained. Using straightforward segmenting tools based upon grayscale differences (the muscle bundles appear much darker, i.e. have lower echogenic echo intensity, than the lighter appearing fibrous tissues), quantitation of the fibrous tissues will be performed using the NIH based freeware ImageJ. Comparisons in muscle fibrous tissues volume will be made with images obtained from subjects with advanced knee joint OA. Static imaging will be performed as a baseline. Additionally, dynamic imaging will be performed with patient co-operation. Passive movement of the joints will also be performed and muscle architecture imaged. The aim of the dynamic imaging is to determine whether the architecture of the fibrous tissue changes during muscle contraction so as to facilitate load bearing as a passive agent. It may be recalled here that nearly 50% cellular content of bulk of a muscle is contributed by cells and fibers of the extracellular matrix.

Thirty (30) randomly chosen patients will receive a 6 month course of doxycycline, alongside conventional therapy. The rest of the patients will receive placebo. At the end of the treatment phase, flexor and extensor groups of muscles around the knee joint will again be imaged, quantitated for fibrous tissue contents by ImageJ, and compared with images obtained from control conditions. Correlation analyses between muscle fibrous tissue contents and circulating TGF-β levels, before and after doxycycline treatment, can be used to show improvement in OA patients. Example 7. OA and inflammatory biomarkers

Tetracycline has been shown to inhibit nitrosothiol production in cytokine simulated osteoarthritis cells. MRI of the knee has shown local cytokine activity that has not been correlated with pain, disability or progression of the disease. These cytokines are involved in the inflammatory phase of degenerative joint diseases. Accordingly, TGF-β levels can be measured in patients with OA of the knee and correlated with cytokine presence on MRI. These patients can be treated with doxycyline for two months. Each patient's TGF-βΙ levels will also be measured monthly. We hypothesize that TGF-β levels will be initially elevated in OA patients, relative to controls lacking OA, and that doxycycline treatment will reduce TGF-β levels over time. Observing a correlation between the severity of OA symptoms (e.g., by global assessment, HOOS, KOOS, and/or WOMAC measures) and TGF-β levels would indicate that TGF-β levels can serve as a useful biomarker in monitoring OA progression and/or responsiveness to treatment.

Example 8. Diagnosis of OA based on level of inflammatory biomarkers

A patient that presents with osteoarthritis or symptoms of osteoarthritis may be assessed for levels of an inflammatory biomarker (e.g., an inflammatory cytokine), such as those shown in Table 1 (e.g., TGF-βΙ , TGF^2, or TGF^3). For example, a blood sample may be drawn from the patient and examined for serum levels of TGF-βΙ . A level of TGF-βΙ in the blood sample that is found to be elevated relative to a standard, such as the normal level of TGF-βΙ shown in Table 1 (0-20 ng/ml), or to the level of TGF-βΙ found in a blood sample from a healthy subject, indicates that the patient is likely to be responsive to treatment with doxycycline. A patient showing such elevated levels of TGF-βΙ can then be treated with doxycycline and monitored for improvement in OA symptoms and for normalization of serum TGF-βΙ levels. The course of doxycycline treatment for such a patient would run for six months. We hypothesize that such doxycycline treatment will improve patient symptoms and, by virtue of its rational targeting of the pro-fibrotic process, significantly ameliorate the pathophysiological processes affecting the different structures (e.g., muscles, synovium, and joints) in osteoarthritis.

Example 9. Acceleration of OA patient response to physical therapy

Eccentric resistance training of the hamstring has been shown to downregulate TGF-βΙ . OA patients will be randomized into placebo versus doxycycline treatment groups to determine if doxycycline can accelerate the response to physical therapy incorporating eccentric resistance. Patients will also be imaged with ultrasound of the hamstring at rest and during exercise. We hypothesize that doxycycline will act synergistically with eccentric resistance training, such that patients undergoing both doxycycline treatment and eccentric resistance training show greater downregulation of TGF-βΙ levels than those undergoing doxycycline treatment or eccentric resistance training alone.

Example 10. Treatment of arthropathy with doxycycline

In this case, a 45 year old female patient presented with inflammatory bowel disease and arthropathy. Control of the disease had been attempted with TNF blockers, but this was unsuccessful despite numerous changes in the therapy. Most recently, the patient was placed on Abatacept (Orencia), but continued to have significant muscle pain and elevations of muscle enzymes. Thus, the patient was placed on a regimen of doxycycline (100 mg doxycycline, once per day). After a month on the doxycycline regimen, the patient noted significant improvement in all symptoms. The patient was subsequently taken off the medication. Two months later, the patient requested that she restart the doxycycline treatment as infusion therapy had continued to fail to provide her with adequate symptom control. The patient is currently back on the 100 mg doxycyline alternate day therapy.

Example 11. Treatment of diabetic adhesive capsulitis with doxycycline

Currently, there is no treatment option for the inflammatory condition of diabetic adhesive capsulitis. A one year monitoring of TGF-βΙ can be performed in patients with adhesive capsulitis and a double blind placebo drug study to look at outcomes in this disease process. Patients will be monitored for elevations in TGF-βΙ . Compounding creams and transdermal administration of doxycycline (100 mg per day for at least 30 days) will be used on affected areas to determine if doxycycline can improve outcomes in adhesive capsulitis. The proposal will include a double blind study after imaging with MRI and ultrasound. The patients will be followed clinically, utilizing a standard 36-question short form quality of life assessment (SF-36) and measurements of range of motion to monitor the functional state of the patient. We will measure levels of TGF-βΙ prior to the treatment arm then monthly throughout the study. It is hypothesized that TGF-βΙ levels will correlate with disease severity and that treatment with doxycycline will significantly improve outcomes and reduce TGF-βΙ levels.

Example 12. Treatment of constipation with doxycyline

Our clinic is currently treating five patients, each suffering from constipation for more than ten years, using submicrobial doses of doxycycline. The clinical symptoms are being monitored by gastroenterology. The gut biome can be monitored, as well, to see if changes are observed as a function unrelated to the antimicrobial affect of the medication. Treatment of the five patients with doxycyline (100 mg per day for at least 30 days) has thus far resulted in 80% improvement in symptoms, indicating that doxycycline treatment can be an effective method for improving outcomes in constipation.

In one case, a 58 year old female patient with presented with rheumatoid arthritis and

constipation. The patient was off all therapies for her rheumatoid arthritis but had persistent leg pain from a bulging disc. The patient was thus placed on a regimen of doxycyline (100 mg daily for 30 days) for leg pain. At the patient's return visit, she reported complete resolution of constipation symptoms. The patient has recently restarted doxycycline treatment for constipation after three months off the drug.

Example 13. Treatment of fibromyalgia with doxycyline

Patients with fibromyalgia can be treated with doxycycline. For each such patient, other diseases can be excluded as the cause of pain, using diagnostic methods known in the art, and can have shown suboptimal responses to current regimens for treating fibromyalgia, such as cognitive behavioral therapy or treatment with pregabalin or duloxetine. Such patients will be placed on a stable regimen of submicrobial doses of doxycycline for at least six months (for example, 100 mg doxycycline per day for at least 30 days). The prescreening may include metabolic assessment, SF-36, a visual analog scale for global pain, monitoring ADL function, and sense of wellbeing. Recruited patients will be treated either with submicrobial doses of doxycycline or placebo and monitored over the course of the study (at least 30 days) for improvement of symptoms. One case involving treatment of fibromyalgia with doxycycline may be presently reported. The patient was a 65 year old woman with a 30 year history of fibromyalgia. This patient required narcotics to manage her pain symptoms and underwent trigger point injections every two months. She became completely disabled from the pain. In July, 2013, the patient was started on a regimen of doxycycline (100 mg daily). On her return visit, she reported feeling normal, requiring no injections and being pain free. Since then she has tried to come off the doxycycline regimen, but each time, all symptoms return after a few months. We have tried alternate regimens, such as three weeks on the doxycycline followed by three weeks of alternate day doxycycline treatment. The patient presently follows up with our clinic every three months.

Example 14. Treatment of exercise intolerance with doxycycline

A 44 year old male patient presented with a three year history of progressive limitation of exercise tolerance. In high school and college, the patient was a sprinter and after school ran regularly up to 10 miles. The patient displayed slight elevations of creatine kinase (CPK ) on laboratory tests, but all other diagnostics were negative. He was evaluated by neurology, orthopedics, and endocrinology without a diagnosis. We placed the patient on a regimen of 100 mg of doxycycline per day for one month. On the patient's first return visit, he has shown improvement to 60 minutes on a treadmill with the ability to lift weights following his cardiovascular workout, representing a significant improvement in the patient's exercise intolerance.

Example 15. Treatment of hypermobility syndrome with doxycycline

In this case, the patient was a 17 year old female with hypermobility syndrome. The patient suffered from joint pain involving hands, knees and feet. Thus, the patient was placed on a regimen of doxycyline (100 mg per day) for three months. As a result, the patient showed complete resolution of all symptoms. After being off the doxycycline regimen for two months, the patient has restarted doxycycline treatment three times weekly due to the return of her joint pain.

Example 16. Treatment of knee inflammation after total knee replacement with doxycycline

Male patient "JS" presented 6 weeks after total knee replacement. JS's first knee replacement two years earlier was without complication. The second replacement was complicated by swelling, limited range of motion, and pain. On examination, his total range of motion was 75 degrees. The knee was warm to touch, swollen and painful. Laboratory tests revealed a normal white blood cell (WBC) count, and his Erythrocyte Sedimentation Rate (ESR) was 1 . Measurement of serum MMP1 level was at 49% of inflammatory threshold seen typically in rheumatoid arthritis. Serum MMP3 levels were at 64% of inflammation scoring for rheumatoid arthritis. JS was placed on 100 mg of doxycyline once daily for three weeks. As a result, he had complete resolution of his swelling. Moreover, his range of motion was now 120 degrees and he was able to ambulate with difficulty. Manipulation of the knee under anesthesia was avoided. The patient has not required further treatment to date. Example 17. Treatment of mouth ulcers, abdominal pain, and low grade fever with doxycycline

Patient "CF" is a 47 year old female with a 24-year history of mouth ulcers, abdominal pain and low grade fevers. She had been evaluated at numerous in-town hospitals without diagnosis or treatment. Her examination was unremarkable, and all laboratory testing was negative with respect to autoimmune diseases. Her Avise profile was negative for autoantibodies, and her ESR and C-reactive protein (CRP) level were within normal range. Her serum MMP1 was at the high end for inflammation, in the 53th- percentile. CF was placed on 50 mg of doxycycline daily, and showed complete resolution of symptoms. After four weeks of therapy, doxycycline was discontinued. All symptoms subsequently recurred, and the patient was placed on alternate-day therapy with 50 mg of doxycycline without relief. The patient is now on 20 mg of Doxycycline daily and will be re-tested for MMP levels after three months of therapy.

Example 18. Treatment of a second fibromyalgia patient with doxycycline

Patient "TP" is a 65 year old white female with a 10-year history of fibromyalgia requiring trigger point injections, pain clinic visits, poor functionality, complete disability. Laboratory testing was unrevealing. The patient was seen every 6-8 weeks for pain management and trigger point injections. Laboratory testing revealed an ESR of 4 and a CRP level of 1 .2. Her serum MMP3 was at the 59th percentile for inflammatory rheumatoid arthritis. TP was placed on submicrobial doses of doxycycline (50 mg daily) and had 80% resolution of symptoms on a visual analog scale (VAS). Her visits have decreased by over 50%. With therapy, her serum MMP3 level in September 2014 was at the 21 th percentile for inflammatory disease associated with rheumatoid arthritis. The patient has been unable to discontinue the medication without resurgence of symptoms and is now on 20 mg of doxycycline daily.

Example 19. Treatment of sarcoidosis with hilar adenopathy with doxycycline

Patient "MC" is a 45 year old white male with a remote history of sarcoidosis with hilar adenopathy. He presented in 2013 with a 5 year history of muscle pain and inability to exercise at his usual level. There were no other contributory symptoms. There was no joint swelling, and muscle examination showed normal strength and no evidence of muscle fasciculations. Neurological examination also showed normal results. MC's laboratory testing was negative for all autoimmune diseases including tests for antinuclear antibodies (ANA), angiotensis-converting enzyme (ACE), rheumatoid arthritis, and normal muscle enzymes. His ESR was 4 and his CRP levels were 3.5. His serum MMP1 level was at 48% of the inflammatory threshold and his serum MMP3 level was at 28% of the inflammatory threshold. The patient failed a treatment course with prednisone and NSAIDS.

However, after a month on doxycycline (100 mg), he was able to resume regular exercise (30 minutes daily on the elliptical). He is continuing on a regimen of 100 mg doxycycline daily with addition of carnitine, Coq10 and citrulline.

Other Embodiments

All publications, patents, and patent applications mentioned in the above specification are hereby incorporated by reference herein in their entirety. Various modifications and variations of the described device and methods of use of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention.

Claims

1 . A method of treating a subject having an elevated TGF-β level associated with an inflammatory condition comprising administering an effective amount of a tetracycline to said subject, wherein said inflammatory condition results from a condition other than bacterial infection.

2. The method of claim 1 , wherein said TGF-β is TGF-βΙ , TGF^2, or TGF^3.

3. The method of claim 1 or 2, wherein said level of TGF-β is determined in a biological sample obtained from said subject.

4. The method of claim 3, wherein said biological sample is or comprises blood.

5. The method of any one of claims 1 -4, wherein said inflammatory condition is selected from the group consisting of mild traumatic brain injury (mTBI), osteoarthritis, fibromyalgia, diabetic adhesive capsulitis, hypermobility syndrome, exercise intolerance, arthropathy, inflammatory bowel disease, constipation, diabetes-associated inflammation, adhesion, Ehlers-Danlos Syndrome, sarcopenia, sarcoidosis, and liver fibrosis.

6. The method of claim 1 , wherein said inflammatory condition is mTBI.

7. The method of claim 1 , wherein said inflammatory condition is osteoarthritis.

8. The method of claim 1 , wherein said inflammatory condition is fibromyalgia.

9. The method of claim 1 , wherein said inflammatory condition is constipation.

10. The method of claim 1 , wherein said inflammatory condition is adhesion.

1 1 . The method of claim 10, wherein said adhesion is the result of injury, surgery, or inflammation.

12. The method of claim 1 1 , wherein said adhesion resulting from surgery is post-operative adhesion after joint replacement surgery.

13. The method of any one of claims 10-12, wherein said adhesion occurs in a joint, tendon, ligament, eye, abdomen, pericardium, or pelvis.

14. The method of claim 13, wherein said joint is a shoulder joint, knee joint, hip joint, ankle joint, elbow joint, or wrist joint.

15. The method of claim 13, wherein said abdominal adhesion is associated with intestinal blockage, bloating, constipation, nausea, vomiting, abdominal pain, or cramps.

16. The method of claim 13, wherein said eye adhesion is associated with glaucoma.

17. A method of treating a subject having an inflammatory condition selected from the group consisting of: mild traumatic brain injury (mTBI), fibromyalgia, diabetic adhesive capsulitis, hypermobility syndrome, exercise intolerance, arthropathy, inflammatory bowel disease, constipation, diabetes-associated inflammation, adhesion, Ehlers-Danlos Syndrome, sarcopenia, sarcoidosis, and liver fibrosis, said method comprising administering an effective amount of a tetracycline to said subject.

18. The method of claim 17, further comprising, prior to said administering step, determining if said subject has said inflammatory condition.

19. The method of claim 17 or 18, wherein said inflammatory condition is mTBI.

20. The method of claim 17 or 18, wherein said inflammatory condition is fibromyalgia.

21 . The method of claim 17 or 18, wherein said inflammatory condition is constipation.

22. The method of claim 17 or 18, wherein said inflammatory condition is adhesion.

23. The method of claim 22, wherein said adhesion is the result of injury, surgery, or inflammation.

24. The method of claim 23, wherein said adhesion resulting from surgery is post-operative adhesion after joint replacement surgery.

25. The method of claim 22, wherein said adhesion occurs in a joint, tendon, ligament, eye, abdomen, pericardium, or pelvis.

26. The method of claim 25, wherein said joint is a shoulder joint, knee joint, hip joint, ankle joint, elbow joint, or wrist joint.

27. The method of claim 25, wherein said abdominal adhesion is associated with intestinal blockage, bloating, constipation, nausea, vomiting, abdominal pain, or cramps.

28. The method of claim 25, wherein said eye adhesion is associated with glaucoma.

29. The method of any one of claims 1 -28, wherein said tetracycline is administered orally, transdermally, topically, intravenously, or by injection.

30. The method of claim 29, wherein said tetracycline is administered orally.

31 . The method of any one of claims 1 -30, wherein a single dose of said tetracycline is administered in an amount between 1 mg and 1000 mg.

32. The method of claim 31 , wherein said dose of said tetracycline administered is an amount between 75 mg and 300 mg.

33. The method of any one of claims 1 -32, wherein said tetracycline is administered at a submicrobial dose.

34. The method of any one of claims 1 -33, wherein said tetracycline is administered one or more times in intervals of 1 -48 hours.

35. The method of claim 34, wherein said tetracycline is administered at least once every 12 hours.

36. The method of claim 34, wherein said tetracycline is administered at least once every 24 hours.

37. The method of any one of claims 1 -36, wherein said tetracycline is administered at least once daily for 1 day to 1 year.

38. The method of claim 37, wherein said tetracycline is administered at least once daily for 1 week to 3 months.

39. The method of claim 38, wherein said tetracycline is administered at least once daily for 1 week.

40. The method of claim 38, wherein said tetracycline is administered at least once daily for 3 months.

41 . The method of any one of claims 1 -40, wherein said tetracycline is selected from the group consisting of doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK-SMA2, tetracycline, chlortetracycline, oxytetracycline, demeclocycline, lymecycline, meclocycline, methacycline,

rolitetracycline, clomocycline, metacycline, pipacycline, incyclinide, and derivatives thereof.

42. The method of claim 1 or 17, wherein said tetracycline is doxycycline.

43. The method of claim 1 or 17, wherein said tetracycline is minocycline.

44. The method of claim 1 or 17, wherein said tetracycline is sarecycline.

45. The method of claim 1 or 17, wherein said tetracycline is omadacycline.

46. The method of claim 42, wherein said doxycycline is administered in an amount of 100 mg at least once daily for at least 30 days.

47. The method of any one of claims 1 -46, further comprising administering a second therapeutic agent.

48. The method of claim 47, wherein said second therapeutic agent is selected from the group consisting of a non-steroidal anti-inflammatory drug (NSAID), a COX-2 inhibitor, capsaicin, and gabapentin.

49. A method for treating an inflammatory condition in a subject, comprising:

(i) determining a level of a TGF-β in a biological sample obtained from said subject, wherein an elevated level of said TGF-β in said biological sample, relative to a level in a normal subject, indicates the presence of said inflammatory condition; and

(ii) administering an effective amount of a tetracycline to said subject if the presence of said inflammatory condition is detected in said subject.

50. The method of claim 49, wherein said TGF-β is TGF-βΙ , TGF^2, or TGF^3.

51 . The method of claim 50, wherein said TGF-β is TGF-βΙ and said level in the normal subject is about 20 ng/ml.

52. The method of any one of claims 49-51 , wherein said tetracycline is administered orally, transdermally, topically, intravenously, or by injection, wherein preferably said tetracycline is administered orally.

53. The method of any one of claims 49-52, wherein a single dose of said tetracycline is administered in an amount between 1 mg and 1000 mg.

54. The method of claim 53, wherein said dose of said tetracycline administered is in an amount between 75 mg and 300 mg.

55. The method of any one of claims 49-54, wherein said tetracycline is administered at a submicrobial dose.

56. The method of any one of claims 49-55, wherein said tetracycline is administered one or more times in intervals of 1 -48 hours.

57. The method of claim 56, wherein said tetracycline is administered at least once every 12 hours.

58. The method of claim 56, wherein said tetracycline is administered at least once every 24 hours.

59. The method of any one of claims 49-58, wherein said tetracycline is administered at least once daily for 1 day to 1 year.

60. The method of claim 59, wherein said tetracycline is administered at least once daily for 1 week to 3 months.

61 . The method of claim 60, wherein said tetracycline is administered at least once daily for 1 week.

62. The method of claim 60, wherein said tetracycline is administered at least once daily for 3 months.

63. The method of claim 49-62, wherein said tetracycline is selected from the group consisting of doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK-SMA2, tetracycline, chlortetracycline, oxytetracycline, demeclocycline, lymecycline, meclocycline, methacycline,

rolitetracycline, clomocycline, metacycline, pipacycline, incyclinide, and a derivative thereof.

64. The method of claim 49, wherein said tetracycline is doxycycline.

65. The method of claim 49, wherein said tetracycline is minocycline.

66. The method of claim 49, wherein said tetracycline is sarecycline.

67. The method of claim 49, wherein said tetracycline is omadacycline.

68. The method of claim 64, wherein said doxycycline is administered in an amount of 100 mg at least once daily for at least 30 days.

69. The method of any of claims 49-68, further comprising administering a second therapeutic agent.

70. The method of claim 69, wherein said second therapeutic agent is selected from the group consisting of a non-steroidal anti-inflammatory drug (NSAID), a COX-2 inhibitor, capsaicin, and gabapentin.

71 . The method of any of claims 49-70, wherein said tetracycline has not previously been administered to said subject to treat said inflammatory condition.

72. The method of any of claims 49-70, wherein said tetracycline has been previously administered to said subject to treat said inflammatory condition.

73. The method of any one of claims 49-72, wherein said biological sample is or comprises blood.

74. The method of any one of claims 49-73, wherein said determining step further comprises determining a level of at least one additional inflammatory biomarker in a biological sample obtained from said subject;

wherein an elevated level of said at least one additional inflammatory biomarker in said biological sample, relative to a level in a normal subject, indicates the presence of said inflammatory condition, and wherein at least one of said additional inflammatory biomarkers is selected from the group consisting of MMP1 , MMP3, MMP9, ILI A, IL1 B, TNFa, IL-6, RANKL, MCP-1 , interferon-gamma, IL-8, CX3CL1 , CXCL1 , CXCL10, SDF-1 , IL-2, IL-17, IL-18, and IL-23.

75. The method of claim 74, wherein said at least one of said additional inflammatory biomarkers is MMP1 and said level in the normal subject is about 30 ng/ml.

76. The method of claim 74, wherein said at least one of said additional inflammatory biomarkers is MMP3 and said level in the normal subject is about 40 ng/ml.

77. The method of claim 74, wherein said at least one of said additional inflammatory biomarkers is IL1 B and said level in the normal subject is about 80 pg/ml.

78. The method of claim 74, wherein said at least one of said additional inflammatory biomarkers is TNFa and said level in the normal subject is about 10 pg/ml.

79. The method of claim 74, wherein said at least one of said additional inflammatory biomarkers is interferon-gamma and said level in the normal subject is about 10 pg/ml.

80. The method of claim 74, wherein said at least one of said additional inflammatory biomarkers is IL1 A and said level in the normal subject is about 5 pg/ml.

81 . The method of claim 74, wherein said at least one of said additional inflammatory biomarkers is IL-6 and said level in the normal subject is about 10 pg/ml.

82. The method of claim 74, wherein said at least one of said additional inflammatory biomarkers is IL-8 and said level in the normal subject is about 10 pg/ml.

83. The method of claim 74, wherein said at least one of said additional inflammatory biomarkers is CX3CL1 and said level in the normal subject is about 1 ng/ml.

84. The method of any of claims 49-83, wherein said inflammatory condition is selected from the group consisting of mTBI, osteoarthritis, fibromyalgia, diabetic adhesive capsulitis, hypermobility syndrome, exercise intolerance, arthropathy, inflammatory bowel disease, constipation, diabetes-associated inflammation, adhesion, Ehlers-Danlos Syndrome, sarcopenia, sarcoidosis, and liver fibrosis.

85. The method of claim 49, wherein said inflammatory condition is mTBI.

86. The method of claim 49, wherein said inflammatory condition is osteoarthritis.

87. The method of claim 49, wherein said inflammatory condition is fibromyalgia.

88. The method of claim 49, wherein said inflammatory condition is constipation.

89. The method of claim 49, wherein said inflammatory condition is adhesion.

90. The method of claim 89, wherein said adhesion is the result of injury, surgery, or inflammation.

91 . The method of claim 90, wherein said adhesion resulting from surgery is post-operative adhesion after joint replacement surgery.

92. The method of claim 89, wherein said adhesion occurs in a joint, tendon, ligament, eye, abdomen, pericardium, or pelvis.

93. The method of claim 92, wherein said joint is a shoulder joint, knee joint, hip joint, ankle joint, elbow joint, or wrist joint.

94. The method of claim 92, wherein said abdominal adhesion is associated with intestinal blockage, bloating, constipation, nausea, vomiting, abdominal pain, or cramps.

95. The method of claim 92, wherein said eye adhesion is associated with glaucoma.

96. A method for treating an inflammatory condition selected from the group consisting of mild traumatic brain injury (mTBI), fibromyalgia, diabetic adhesive capsulitis, hypermobility syndrome, exercise intolerance, arthropathy, inflammatory bowel disease, constipation, diabetes-associated inflammation, adhesion, Ehlers-Danlos Syndrome, sarcopenia, sarcoidosis, and liver fibrosis, in a subject, comprising:

(i) determining a level of an inflammatory biomarker, wherein an elevated level of said inflammatory biomarker in said biological sample, relative to a level in a normal subject, indicates the presence of said inflammatory condition; and

97. The method of claim 96, wherein said inflammatory biomarker is MMP1 and said level in the normal subject is about 30 ng/ml.

98. The method of claim 96, wherein said inflammatory biomarker is MMP3 and said level in the normal subject is about 40 ng/ml.

99. The method of claim 96, wherein said inflammatory biomarker is IL1 B and said level in the normal subject is about 80 pg/ml.

100. The method of claim 96, wherein said inflammatory biomarker is TNFa and said level in the normal subject is about 10 pg/ml.

101 . The method of claim 96, wherein said inflammatory biomarker is interferon-gamma and said level in the normal subject is about 10 pg/ml.

102. The method of claim 96, wherein said inflammatory biomarker is IL1 A and said level in the normal subject is about 5 pg/ml.

103. The method of claim 96, wherein said inflammatory biomarker is IL-6 and said level in the normal subject is about 10 pg/ml.

104. The method of claim 96, wherein said inflammatory biomarker is IL-8 and said level in the normal subject is about 10 pg/ml.

105. The method of claim 96, wherein said inflammatory biomarker is CX3CL1 and said level in the normal subject is about 1 ng/ml.

106. The method of any one of claims 96-105, wherein said inflammatory condition is mTBI.

107. The method of any one of claims 96-105, wherein said inflammatory condition is fibromyalgia.

108. The method of any one of claims 96-105, wherein said inflammatory condition is constipation.

109. The method of any one of claims 96-105, wherein said inflammatory condition is adhesion.

1 10. The method of claim 109, wherein said adhesion is the result of injury, surgery, or inflammation.

1 1 1 . The method of claim 1 10, wherein said adhesion resulting from surgery is post-operative adhesion after joint replacement surgery.

1 12. The method of any one of claims 109-1 1 1 , wherein said adhesion occurs in a joint, tendon, ligament, eye, abdomen, pericardium, or pelvis.

1 13. The method of claim 1 12, wherein said joint is a shoulder joint, knee joint, hip joint, ankle joint, elbow joint, or wrist joint.

1 14. The method of claim 1 12, wherein said abdominal adhesion is associated with intestinal blockage, bloating, constipation, nausea, vomiting, abdominal pain, or cramps.

1 15. The method of claim 1 12, wherein said eye adhesion is associated with glaucoma.

1 16. The method of any one of claims 1 -1 15, wherein said subject is human.

1 17. A kit for treating an inflammatory condition, comprising a device for detecting a level of at least one inflammatory biomarker in a biological sample, a tetracycline, and instructions for use of said tetracycline in a subject in need thereof, wherein at least one of said inflammatory biomarkers is a TGF-β.

1 18. The kit of claim 1 17, wherein said TGF-β is TGF-βΙ , TGF^2, or TGF^3.

1 19. The kit of claim 1 17 or 1 18, wherein said instructions specify use of an effective amount of said tetracycline in said subject if a level of at least one of the inflammatory biomarkers is determined, using said device, to be elevated relative to a level of the same inflammatory biomarker in a normal subject.

120. The kit of any one of claims 1 17-1 19, wherein said inflammatory condition results from a condition other than bacterial infection.

121 . The kit of claim 120, wherein said inflammatory condition is selected from the group consisting of mTBI, osteoarthritis, fibromyalgia, diabetic adhesive capsulitis, hypermobility syndrome, exercise intolerance, arthropathy, inflammatory bowel disease, constipation, diabetes-associated inflammation, adhesion, Ehlers-Danlos Syndrome, sarcopenia, sarcoidosis, and liver fibrosis.

122. The kit of claim 1 17, wherein said inflammatory condition is mTBI.

123. The kit of claim 1 17, wherein said inflammatory condition is osteoarthritis.

124. The kit of claim 1 17, wherein said inflammatory condition is fibromyalgia.

125. The kit of claim 1 17, wherein said inflammatory condition is constipation.

126. The kit of claim 1 17, wherein said inflammatory condition is adhesion .

127. The kit of claim 126, wherein said adhesion is the result of injury, surgery, or inflammation.

128. The kit of claim 127, wherein said adhesion resulting from surgery is post-operative adhesion after joint replacement surgery.

129. The kit of any one of claims 126-128, wherein said adhesion occurs in a joint, tendon, ligament, eye, abdomen, pericardium, or pelvis.

130. The kit of claim 129, wherein said joint is a shoulder joint, knee joint, hip joint, ankle joint, elbow joint, or wrist joint.

131 . The kit of claim 129, wherein said abdominal adhesion is associated with intestinal blockage, bloating, constipation, nausea, vomiting, abdominal pain, or cramps.

132. The kit of claim 129, wherein said eye adhesion is associated with glaucoma.

133. The kit of any one of claims 1 17-132, wherein at least one of said inflammatory biomarkers is selected from the group consisting of MMP1 , MMP3, MMP9, ILI A, IL1 B, TNFa, IL-6, RANKL, MCP-1 , interferon-gamma, IL-8, CX3CL1 , CXCL1 , CXCL10, SDF-1 , IL-2, IL-17, IL-18, and IL-23.

134. The kit of claim 133, wherein said instructions specify that when said TGF-β is TGF-βΙ , detection of a level of said TGF-βΙ above about 20 ng/ml indicates the presence of said inflammatory condition.

135. The kit of claim 133, wherein said instructions specify that when said at least one of said inflammatory biomarkers is MMP1 , detection of a level of said MMP1 above about 30 ng/ml indicates the presence of said inflammatory condition.

136. The kit of claim 133, wherein said instructions specify that when said at least one of said inflammatory biomarkers is MMP3, detection of a level of said MMP3 above about 40 ng/ml indicates the presence of said inflammatory condition.

137. The kit of claim 133, wherein said instructions specify that when said at least one of said inflammatory biomarkers is IL1 B, detection of a level of said IL1 B above about 80 pg/ml indicates the presence of said inflammatory condition.

138. The kit of claim 133, wherein said instructions specify that when said at least one of said inflammatory biomarkers is TNFa, detection of a level of said TNFa above about 10 pg/ml indicates the presence of said inflammatory condition.

139. The kit of claim 133, wherein said instructions specify that when said at least one of said inflammatory biomarkers is interferon-gamma, detection of a level of said interferon-gamma above 10 about pg/ml indicates the presence of said inflammatory condition.

140. The kit of claim 133, wherein said instructions specify that when said at least one of said inflammatory biomarkers is IL1 A, detection of a level of said IL1 A above about 5 pg/ml indicates the presence of said inflammatory condition.

141 . The kit of claim 133, wherein said instructions specify that when said at least one of said inflammatory biomarkers is IL-6, detection of a level of said IL-6 above about 10 pg/ml indicates the presence of said inflammatory condition.

142. The kit of claim 133, wherein said instructions specify that when said at least one of said inflammatory biomarkers is IL-8, detection of a level of said IL-8 above about 10 pg/ml indicates the presence of said inflammatory condition.

143. The kit of claim 133, wherein said instructions specify that when said at least one of said inflammatory biomarkers is CX3CL1 , detection of a level of said CX3CL1 above about 1 ng/ml indicates the presence of said inflammatory condition.

144. The kit of any one of claims 1 17-143, wherein said tetracycline is selected from the group consisting of doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK-SMA2, tetracycline, chlortetracycline, oxytetracycline, demeclocycline, lymecycline, meclocycline, methacycline,

145. The kit of claim 1 17, wherein said tetracycline is doxycycline.

146. The kit of claim 1 17, wherein said tetracycline is minocycline.

147. The kit of claim 1 17, wherein said tetracycline is sarecycline.

148. The kit of claim 1 17, wherein said tetracycline is omadacycline.

149. The method of any one of claims 96-1 16, wherein said tetracycline is selected from the group consisting of doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK-SMA2, tetracycline, chlortetracycline, oxytetracycline, demeclocycline, lymecycline, meclocycline, methacycline, rolitetracycline, clomocycline, metacycline, pipacycline, incyclinide, and a derivative thereof.

150. The method of claim 96, wherein said tetracycline is doxycycline.

151 . The method of claim 96, wherein said tetracycline is minocycline.

152. The method of claim 96, wherein said tetracycline is sarecycline.

153. The method of claim 96, wherein said tetracycline is omadacycline.