WO2015153864A2 - Méthodes de traitement d'états inflammatoires - Google Patents

Méthodes de traitement d'états inflammatoires Download PDF

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WO2015153864A2
WO2015153864A2 PCT/US2015/024047 US2015024047W WO2015153864A2 WO 2015153864 A2 WO2015153864 A2 WO 2015153864A2 US 2015024047 W US2015024047 W US 2015024047W WO 2015153864 A2 WO2015153864 A2 WO 2015153864A2
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inflammatory
tetracycline
level
adhesion
subject
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PCT/US2015/024047
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WO2015153864A3 (fr
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Patricia T. HOPKINS
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Hopkins Patricia T
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/65—Tetracyclines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Definitions

  • the present invention relates to treating inflammatory conditions in subjects, such as mild traumatic brain injury (mTBI) and osteoarthritis.
  • subjects such as mild traumatic brain injury (mTBI) and osteoarthritis.
  • 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 mild traumatic brain injury
  • 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.
  • mTBI 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.
  • CTE chronic traumatic encephalopathy
  • 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.
  • immunoexcitotoxicity is a primary basis for disease pathophysiology (Patterson and Holahan, Front. Cell Neurosci., 6(58): 1 -10, 2012).
  • CSF cerebral spastic syndrome
  • PTSD post-traumatic stress disorder
  • Osteoarthritis 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).
  • the invention features methods and kits for treating subjects with inflammatory conditions, as well as methods for detecting inflammatory conditions.
  • 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
  • mTBI mild traumatic brain injury
  • OA osteoarthritis
  • fibromyalgia diabetic adhesive capsulitis
  • hypermobility syndrome exercise intolerance
  • arthropathy inflammatory bowel disease
  • constipation constipation
  • adhesion e.g., post-operative adhesion, post-operative adhesion after joint replacement surgery, or abdominal adhesion
  • the adhesion is the result of injury, surgery (e.g., joint replacement surgery), or inflammation.
  • the adhesion occurs in a joint, tendon, ligament, eye, abdomen, pericardium, or pelvis.
  • the joint is a shoulder joint, knee joint, hip joint, ankle joint, elbow joint, or wrist joint.
  • 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.
  • the adhesion occurs in the eye (e.g., an eye adhesion) and is further associated with glaucoma.
  • the invention features a method of treating a subject having an elevated TGF- ⁇ 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.
  • the subject has an elevated TGF- ⁇ level (e.g., TGF- ⁇ ,
  • the TGF- ⁇ is TGF- ⁇ .
  • the level of TGF- ⁇ 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).
  • the biological sample is or includes blood.
  • 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- ⁇ , TGF ⁇ 2, or TGF ⁇ 3; preferably TGF- ⁇ ) 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).
  • 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), synovi
  • Inflammatory biomarkers e.g., cytokines
  • Table 1 Inflammatory biomarkers 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.
  • the subject has a sterile inflammation.
  • 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.
  • the inflammatory condition may be mTBI, OA, fibromyalgia, constipation, or adhesion.
  • the adhesion is the result of injury, surgery (e.g., joint replacement surgery), or inflammation.
  • the adhesion occurs in a joint, tendon, ligament, eye, abdomen, pericardium, or pelvis.
  • the joint is a shoulder joint, knee joint, hip joint, ankle joint, elbow joint, or wrist joint.
  • 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.
  • the adhesion occurs in the eye (e.g., an eye adhesion) and is further associated with glaucoma.
  • 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;
  • 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.
  • 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).
  • 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).
  • the adhesion is the result of injury, surgery (e.g., joint replacement surgery), or inflammation.
  • the adhesion occurs in a joint, tendon, ligament, eye, abdomen, pericardium, or pelvis.
  • the joint is a shoulder joint, knee joint, hip joint, ankle joint, elbow joint, or wrist joint.
  • 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.
  • the adhesion occurs in the eye (e.g., an eye adhesion) and is further associated with glaucoma.
  • 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.
  • the tetracycline is administered orally.
  • the tetracycline is doxycycline that is administered orally.
  • the tetracycline is doxycycline that is administered transdermally.
  • the tetracycline is doxycycline that is administered topically.
  • 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.
  • the 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
  • the tetracycline is administered at a submicrobial dose.
  • 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.
  • doxycycline e.g., doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK-SMA2,
  • 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.
  • 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.
  • the tetracycline is administered at least once daily for about 1 week, 1 month (30 days), or 3 months.
  • 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.
  • the tetracycline is doxycycline, minocycline, sarecycline, or omadacycline.
  • the tetracycline is doxycycline or sarecycline.
  • doxycycline is administered in an amount of 100 mg at least once daily for at least 30 days.
  • the method further involves administering a second therapeutic agent.
  • 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.
  • 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- ⁇ , TGF ⁇ 2, or TGF ⁇ 3), in a biological sample taken from the subject.
  • TGF- ⁇ , TGF ⁇ 2, or TGF ⁇ 3 e.g., TGF- ⁇ , TGF ⁇ 2, or TGF ⁇ 3
  • the invention features a method for treating an inflammatory condition in a subject by determining a level of a TGF- ⁇ (e.g., TGF- ⁇ , TGF ⁇ 2, or TGF ⁇ 3) in a biological sample taken from the subject.
  • an effective amount of a tetracycline e.g., doxycycline, minocycline, sarecycline, or omadacycline; preferably doxycycline or sarecycline
  • a tetracycline e.g., doxycycline, minocycline, sarecycline, or omadacycline; preferably doxycycline or sarecycline
  • the TGF- ⁇ is TGF- ⁇ , TGF ⁇ 2, or TGF ⁇ 3.
  • the TGF- ⁇ is TGF- ⁇ and the level in the normal subject is about 20 ng/ml.
  • 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.
  • 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.
  • the 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
  • 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.
  • the tetracycline is administered at a submicrobial dose.
  • 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.
  • 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,
  • 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.
  • 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.
  • 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.
  • the tetracycline is doxycycline, minocycline, sarecycline, or omadacycline.
  • the tetracycline is doxycycline or sarecycline.
  • doxycycline is administered in an amount of 100 mg at least once daily for at least 30 days.
  • 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).
  • NSAID non-steroidal anti-inflammatory drug
  • COX-2 inhibitor e.g., a COX-2 inhibitor
  • capsaicin e.g., a gabapentin
  • the tetracycline has not previously been administered to the subject to treat the inflammatory condition. In other embodiments, the tetracycline has been previously been administered to the subject to treat the inflammatory condition. In other embodiments, the tetracycline has been previously been administered to the subject to treat the inflammatory condition.
  • the biological sample is or comprises blood.
  • 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.
  • a level of at least one additional inflammatory biomarker e.g., an inflammatory biomarker listed in Table 1
  • 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.
  • a level of the inflammatory biomarker greater than that considered to be a normal level for that biomarker indicates the presence of an inflammatory condition.
  • a level of the inflammatory biomarker greater than that corresponding to mild, intermediate, or high inflammation for that biomarker indicates the presence of an inflammatory condition.
  • 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.
  • the at least one of the additional inflammatory biomarkers is interferon-gamma and the level in the normal subject is ⁇ 10 pg/ml.
  • the at least one of the additional inflammatory biomarkers is IL1 A and the level in the normal subject is ⁇ 5 pg/ml.
  • the at least one of the additional inflammatory biomarkers is IL-6 and the level in the normal subject is ⁇ 10 pg/ml.
  • the at least one of the additional inflammatory biomarkers is IL-8 and the level in the normal subject is ⁇ 10 pg/ml.
  • the at least one of the additional inflammatory biomarkers is CX3CL1 and the level in the normal subject is ⁇ 1 ng/ml.
  • 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.
  • 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.
  • adhesion e.g., post-operative adhesion, post- operative adhesion after joint replacement surgery, or abdominal adhesion
  • constipation e.g., post-operative adhesion, post- operative adhesion after joint replacement surgery, or abdominal adhesion
  • constipation e.g., constipation, diabetes- associated inflammation, Ehlers-Danlos Syndrome, sarcopenia, sarcoidosis, and liver fibrosis.
  • 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).
  • adhesion is the result of injury, surgery (e.g., joint replacement surgery), or inflammation.
  • the adhesion occurs in a joint, tendon, ligament, eye, abdomen, pericardium, or pelvis.
  • the joint is a shoulder joint, knee joint, hip joint, ankle joint, elbow joint, or wrist joint.
  • 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.
  • the adhesion occurs in the eye (e.g., an eye adhesion) and is further associated with glaucoma.
  • 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:
  • 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;
  • the invention features a method of treating a subject, in which the following steps are performed:
  • determining a level of at least one inflammatory biomarker e.g., a cytokine
  • 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- ⁇ is TGF- ⁇ , 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.
  • the invention features a method for treating an inflammatory condition in a subject by:
  • determining a level of at least one inflammatory biomarker e.g., a cytokine
  • a cytokine such as those shown in Table 1 (e.g., TGF- ⁇ , TGF ⁇ 2, TGF ⁇ 3, MMP1 , or MMP3; preferably TGF- ⁇ )
  • TGF- ⁇ a cytokine
  • MMP1 a inflammatory biomarker
  • 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- ⁇ is TGF- ⁇ , the level in a normal subject can be about 20 ng/ml), indicates the presence of the inflammatory condition; and
  • 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
  • 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
  • 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.
  • 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
  • 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.
  • 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.
  • adhesion e.g., post-operative adhesion, post-operative adhesion after joint replacement surgery, or abdominal adhesion
  • constipation e.g., constipation, diabetes- associated inflammation, Ehlers-Danlos Syndrome, sarcopenia, sarcoidosis, and liver fibrosis.
  • 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).
  • adhesion is the result of injury, surgery (e.g., joint replacement surgery), or inflammation.
  • the adhesion occurs in a joint, tendon, ligament, eye, abdomen, pericardium, or pelvis.
  • the joint is a shoulder joint, knee joint, hip joint, ankle joint, elbow joint, or wrist joint.
  • 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.
  • the adhesion occurs in the eye (e.g., an eye adhesion) and is further associated with glaucoma.
  • the inflammatory condition results from a condition other than bacterial infection.
  • the subject has a sterile inflammation.
  • the tetracycline e.g., doxycycline, minocycline, or another tetracycline disclosed herein; preferably doxycycline or sarecycline
  • the tetracycline is administered orally, transdermally, topically, intravenously, or by injection.
  • 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.
  • 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.
  • the tetracycline is administered at a submicrobial dose.
  • 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.
  • the tetracycline is administered at least once every 12 or 24 hours.
  • 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.
  • 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.
  • the tetracycline is administered at least once daily for 1 week, 1 month (30 days), or 3 months.
  • 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.
  • the tetracycline is doxycycline, minocycline, sarecycline, or omadacycline.
  • the tetracycline is doxycycline, minocycline, or sarecycline.
  • doxycycline is administered in an amount of 100 mg at least once daily for at least 30 days.
  • the method further involves administering a second therapeutic agent.
  • the second therapeutic agent is selected from the group consisting of a nonsteroidal anti-inflammatory drug (NSAID), a COX-2 inhibitor, capsaicin, and gabapentin.
  • the tetracycline has not previously been administered to the subject to treat the inflammatory condition.
  • the tetracycline has been previously administered to the subject to treat the inflammatory condition.
  • the biological sample is or comprises blood.
  • the inflammatory biomarker e.g., a cytokine
  • the inflammatory biomarker is selected from the group consisting of TGF- ⁇ , 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- ⁇ .
  • the inflammatory biomarker is TGF- ⁇ 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.
  • 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.
  • 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.
  • the inflammatory condition is mTBI, OA, or fibromyalgia.
  • the subject is human.
  • 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.
  • 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).
  • an injury e.g., a head injury and/or combat injury.
  • the subject has suffered multiple injuries (e.g., repetitive head injuries and/or repetitive brain injuries).
  • 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- ⁇ , TGF ⁇ 2, TGF ⁇ 3, MMP1 , or MMP3; preferably TGF- ⁇ ) 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.
  • the inflammatory biomarker is a TGF- ⁇ (e.g.
  • 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.
  • the inflammatory biomarker is TGF- ⁇ and the elevated level is at least about 20 ng/ml.
  • the inflammatory condition results from a condition other than bacterial infection.
  • the subject has a sterile inflammation.
  • 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.
  • adhesion e.g., post-operative adhesion, post-operative adhesion after joint replacement surgery, or abdominal adhesion
  • Ehlers-Danlos Syndrome sarcopenia, sarcoidosis, and liver fibrosis.
  • the inflammatory condition is mTBI, OA, fibromyalgia, constipation, or adhesion (e.g., postoperative adhesion, post-operative adhesion after joint replacement surgery, or abdominal adhesion).
  • adhesion is the result of injury, surgery (e.g., joint replacement surgery), or inflammation.
  • the adhesion occurs in a joint, tendon, ligament, eye, abdomen, pericardium, or pelvis.
  • the joint is a shoulder joint, knee joint, hip joint, ankle joint, elbow joint, or wrist joint.
  • 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.
  • the adhesion occurs in the eye (e.g., an eye adhesion) and is further associated with glaucoma.
  • At least one of the inflammatory biomarkers is selected from the group consisting of TGF- ⁇ , 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • the tetracycline is doxycycline, minocycline, sarecycline, omadacycline, PTK-RA01 , PTK-MS01 , PTK-SMA2, tetracycline,
  • 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:
  • R 1 is hydrogen, optionally substituted amino, or halo
  • R 2 and R 3 are independently hydrogen, hydroxy, or optionally substituted amino
  • R 4 and R 5 are independently hydrogen, optionally substituted CrC 6 alkyl, or optionally substituted C 2 -Cg heterocyclyl CrC 6 alkyl;
  • R 6 is hydroxy or -CH 2 NR 12 R 13 ;
  • R 7 is hydrogen, cyano, optionally substituted C Ce alkyl, optionally substituted ( Ce alkynyl, optionally substituted C 6 -C 10 aryl, or -CH 2 NR 14 R 15 ;
  • R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 ,and R 15 are independently hydrogen, hydroxy, optionally substituted C Ce alkyl, or optionally substituted C Ce alkoxy;
  • R 1 is hydrogen, -N(CH 3 ) 2 , or halo;
  • R 2 and R 3 are independently hydrogen, hydroxy, or -N(CH 3 ) 2 ;
  • R is hydrogen, * * ⁇ , ,
  • R 7 is hydrogen
  • R 8 , R 9 , R 10 , 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:
  • 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.
  • EULAR European League against Rheumatism
  • 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.
  • 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
  • alkyl 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.
  • cycloalkyl 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.
  • 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.
  • 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).
  • 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.
  • any hydrogen atom on one of these groups can be replaced with a substituent as described herein.
  • the term "aminoalkyl” refers to an alkyl group, as defined herein, comprising an optionally substituted amino group (e.g., NH 2 ).
  • 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.
  • 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.
  • heteroatom is O or N.
  • heterocyclyl 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.
  • 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.
  • 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.
  • 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.
  • heterocyclylalkyl thus includes a heterocyclic group as defined above connected to an alkyl, heteroalkyi, alkenyl, heteroalkenyl, alkynyl or heteroalkynyl moiety also as defined above.
  • 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.
  • 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.
  • heteroalkenyl or heteroalkynyl when 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.
  • heteroalkyi, heteroalkenyl and heteroalkynyl groups include CH 2 OCH 3 , CH 2 N(CH 3 ) 2 , CH 2 OH, (CH 2 ) n NR 2 , OR, COOR, CONR 2 , (CH 2 ) n OR,(CH 2 ) n COR, (CH 2 ) n COOR, (CH 2 ) n SR, (CH 2 ) n SOR,
  • alkylene alkenylene
  • alkynylene alkynylene
  • alk 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).
  • saturated groups e.g., alkylene or alk
  • C2-C3, C2-C4, C2-C6, or C2-C8 unsaturated groups
  • alkaryl represents an aryl group, as defined herein, attached to the parent molecular group through an alkylene group, as defined herein
  • 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.
  • heteroalkylene, heteroalkenylene or heteroalkynylene group replaces one carbon atom of the alkylene, alkenylene or alkynylene group to which the heteroform corresponds.
  • these heteroforms do not contain more than three contiguous heteroatoms.
  • 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.
  • R is an optionally substituted alkyi group (e.g., C1 -C6 alkyi group), unless otherwise specified.
  • 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.
  • 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.
  • 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.
  • amino represents -N(R N1 ) 2 , wherein each R N1 is, independently, H, OH, N0 2 , N(R N2 ) 2 , S0 2 OR N2 , S0 2 R 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.
  • amino is -NH 2 , or -NHR N1 , wherein R N1 is, independently, OH, N0 2 , NH 2 , NR N2 2 , S0 2 OR N2 , S0 2 R N2 , SOR N2 , alkyi, or aryl, and each R N2 can be H, alkyi, or aryl.
  • R N1 is, independently, OH, N0 2 , NH 2 , NR N2 2 , S0 2 OR N2 , S0 2 R N2 , SOR N2 , alkyi, or aryl
  • each R N2 can be H, alkyi, or aryl.
  • aminoalkyl 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
  • Aromatic moiety or aryl 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.
  • 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.
  • the ring systems contain 5 12 ring member atoms or 6-10 ring member atoms.
  • 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.
  • 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.
  • 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.
  • haloalkyl 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.
  • the haloalkyl group can be further substituted with 1 , 2, 3, or 4 substituent groups as described herein for alkyl groups.
  • hydroxy represents an -OH group.
  • hydroxyalkyl 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.
  • V-protecting group 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, isopropyloxycarbony
  • 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).
  • a "thiol” group is a substituent have the structure -S-H.
  • Typical optional substituents on aromatic or heteroaromatic groups include independently halo, CN, N0 2 , CF 3 , OCF 3 , COOR', CONR' 2 , OR', SR", SOFT, S0 2 R', NR' 2 , NR'(CO)R',NR'C(0)OR',
  • 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.
  • non-aromatic groups e.g., alkyl, alkenyl, and alkynyl groups
  • a substituent group e.g., alkyl, alkenyl, alkynyl, or aryl (including all heteroforms defined above) may itself optionally be substituted by additional substituents.
  • additional substituents e.g., alkyl, alkenyl, alkynyl, or aryl (including all heteroforms defined above
  • 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.
  • alkyl substituted by aryl, amino, halo and the like would be included.
  • the group may be substituted with 1 , 2, 3, 4, 5, or 6 substituents.
  • 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.
  • 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 NH 2 (CH 2 ) n COOH 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.
  • 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 NH 2 (CH 2 ) n COOH wherein n is 2-6, neutral nonpolar amino acids, such as sarcosine
  • inflammatory condition refers to any disease, disorder, or condition associated with an inflammatory response.
  • 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.
  • 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.
  • 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.
  • 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).
  • a non-bacterial infective organism e.g., a fungus, virus, or protist
  • 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.
  • 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).
  • microbial e.g., bacterial
  • 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.
  • 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).
  • symptoms of inflammation include: altered body temperature (e.g., less than
  • increased heart rate e.g., greater than 90 beats per minute
  • tachypnea e.g., greater than 20 breaths per minute
  • decreased arterial pressure of C0 2 e.g., less than 4.3 kPa
  • altered white blood count e.g., less than 4,000 cells/mm 3 or greater than 12,000 cells/mm 3
  • 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.
  • diabetes-associated inflammation 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.
  • extracellular matrix crosslinking and/or fibrosis in tissues such as adipose tissue, the liver, pancreatic islets, and the vasculature.
  • 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 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).
  • biological sample body fluid sample
  • sample 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.
  • 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.
  • 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.
  • 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.
  • an inflammatory biomarker such as a cytokine (e.g., a TGF- ⁇ )
  • cytokine e.g., a TGF- ⁇
  • an inflammatory biomarker such as a cytokine (e.g., a TGF- ⁇ )
  • TGF- ⁇ e.g., a TGF- ⁇
  • TGF- ⁇ a simple early diagnosis or differential diagnosis can be achieved.
  • the progress of therapy can be monitored more easily by testing such biological samples for target genes or gene products.
  • the prediction of outcome or response to therapy can be tested more easily by testing such biological samples for target genes or gene products.
  • biomarker refers 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- ⁇ , TGF ⁇ 2, or TGF ⁇ 3).
  • TGF- ⁇ is meant a member of the TGF- ⁇ gene family or a polypeptide encoded by a gene in the TGF- ⁇ family.
  • Members of the TGF- ⁇ family include, for example, TGF- ⁇ , TGF ⁇ 2, and TGF ⁇ 3.
  • 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.
  • an "effective amount" of an agent 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.
  • 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).
  • 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
  • antimicrobial activity refers to clinically relevant antimicrobial activity.
  • Submicrobial doses include, for example, doses below 200
  • 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.
  • 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).
  • course of therapy is meant the length of time from the initiation of a therapy until the conclusion of the therapy.
  • 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 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
  • 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.
  • 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 adhe
  • 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).
  • an elevated or “elevated level” is meant that the expression level of a biomarker (e.g., an inflammatory biomarker such as a TGF- ⁇ ) 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).
  • 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).
  • normal subject 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).
  • 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- ⁇ , TGF ⁇ 2, or TGF ⁇ 3; preferably TGF- ⁇ ), is within the "normal level.”
  • a level of an inflammatory biomarker e.g., one or more of the inflammatory biomarkers listed in Table 1 (e.g., TGF- ⁇ , TGF ⁇ 2, or TGF ⁇ 3; preferably TGF- ⁇ ) is within the "normal level.”
  • 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- ⁇ relative to a level in a normal subject).
  • 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.
  • 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
  • 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
  • inflammatory conditions e.g., those caused by head trauma, such as mTBI, or other sterile inflammatory conditions
  • inflammatory biomarkers e.g., cytokines, such as TGF- ⁇
  • Methods for detecting an inflammatory condition in a subject involve, for example, the determination of a level of at least one inflammatory biomarker (e.g., a cytokine, such as TGF- ⁇ ) in a biological sample obtained from the subject (e.g., blood or cerebrospinal fluid).
  • a level of at least one inflammatory biomarker e.g., a cytokine, such as TGF- ⁇
  • a biological sample obtained from the subject e.g., blood or cerebrospinal fluid.
  • 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).
  • 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.
  • an inflammatory biomarker e.g., a cytokine, such as TGF- ⁇
  • 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 d
  • 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).
  • a level of at least one inflammatory biomarker such as the inflammatory biomarkers shown in Table 1
  • 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).
  • 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.
  • 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.
  • 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.
  • a inflammatory biomarker e.g., an inflammatory cytokine
  • 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.
  • the present invention features methods and kits for detecting the presence of an inflammatory condition in a subject.
  • 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.
  • 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.
  • the inflammatory conditions may be mTBI, OA, fibromyalgia, constipation, or an adhesion as described herein.
  • 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 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.
  • 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
  • tissue inflammation e.g., symptoms such as localized pain, redness, or swelling
  • 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).
  • 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.
  • 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.
  • 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:
  • mTBI 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.
  • 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).
  • the subject exposed to head trauma may exhibit extracranial injuries.
  • 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.
  • the subject exposed to a head trauma may exhibit only superficial external injuries or no extra-cranial injuries.
  • 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.
  • the brain a soft mass surrounded by fluid that allows it to "float,” may rebound against the skull resulting in further damage.
  • 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").
  • ICP intracranial pressure
  • 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.
  • the determination of whether the subject has suffered mTBI can be completed immediately following a head trauma, or at any time thereafter.
  • 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 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.
  • 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 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.
  • 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;
  • OA osteoarthritis
  • 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.
  • ECM extracellular matrix
  • 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- ⁇ and TNF-a.
  • pro-inflammatory biomarkers e.g., pro-inflammatory cytokines
  • 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.
  • osteoarthritis diagnostic and monitoring methods 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.
  • HOOS Hip Disability and Osteoarthritis Outcome Score
  • KOOS Knee Injury and Osteoarthritis Outcome Score
  • WOMAC Western Ontario McMaster University Osteoarthritis Index
  • 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.
  • 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.
  • 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
  • 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).
  • 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.
  • 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
  • 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:
  • 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.
  • 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.
  • 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.
  • 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.
  • immunoassay format e.g., ELISA, Western blot, or RIA assay
  • Inflammatory biomarkers whose levels may be usefulness in the diagnosis and monitoring of such inflammatory conditions include, but are not limited to: TGF- ⁇ , 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- ⁇ , TGF ⁇ 2, or TGF ⁇ 3; most preferably TGF- ⁇ ).
  • Expression levels of a TGF- ⁇ and one or more additional inflammatory biomarkers 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.
  • 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- ⁇ , 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.
  • an inflammatory biomarker e.g., an inflammatory cytokine
  • the cytokine detected is TGF- ⁇ , TGF ⁇ 2, or TGF ⁇ 3; most preferably TGF- ⁇ .
  • 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.
  • inflammatory biomarkers e.g., TGF- ⁇
  • TGF- ⁇ 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.
  • TGF- ⁇ 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 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.
  • proteins or nucleic acids e.g., cytokines, downstream effectors of one or more cytokines, or enzymatic activity (for example, matrix metalloproteinase activity)
  • enzymatic activity for example, matrix metalloproteinase activity
  • 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.
  • 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.
  • 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
  • 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 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.
  • a fixative is determined by the purpose for which the sample is to be histologically stained or otherwise analyzed.
  • neutral buffered formalin, Bouin's or paraformaldehyde may be used to fix a sample.
  • 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.
  • 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.
  • 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.
  • 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.
  • in situ hybridization for example, fluorescence in situ hybridization (FISH) (see, e.g., Angerer et al., Methods Enzymol. 152: 649- 661 , 1987).
  • FISH fluorescence in situ hybridization
  • 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 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.
  • 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.
  • bead based multiplex assays such as Luminex
  • MRM multiple reaction monitoring
  • Amplification-based assays also can be used to measure the expression level of one or more genes.
  • 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).
  • PCR polymerase chain reaction
  • 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.
  • the 5' nuclease activity of the polymerase for example, AmpliTaq
  • LCR ligase chain reaction
  • 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.
  • microarray-based platforms e.g., single- nucleotide polymorphism (SNP) arrays
  • RNA-Seq next generation sequencing platforms
  • 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).
  • 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 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.
  • 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
  • primers and probes comprising the sequences described herein are used to detect expression of one or more genes, as described herein.
  • 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).
  • 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.
  • mRNAs such as target mRNAs
  • 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.
  • 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.
  • a selected gene or biomarker in a tissue or cell sample and/or the activity of a gene or gene product of interest may be examined by way of functional or activity-based assays.
  • the biomarker is an enzyme
  • a non-limiting example of a biological activity that may be measured to determine TGF- ⁇ 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.
  • the solid supported can be adapted to allow for automation of any one of the methods described herein (e.g., PCR).
  • 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).
  • a condition e.g., an inflammatory condition, such as mTBI, OA, or fibromyalgia.
  • 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.
  • 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
  • the inflammatory condition is mTBI, OA, fibromyalgia, constipation, or adhesion.
  • the subject may have, for example, an elevated level of at least one TGF- ⁇ (e.g., TGF- ⁇ , 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- ⁇ ).
  • 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- ⁇ ).
  • 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.
  • 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
  • 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- ⁇ , in the subject, according to the levels of inflammation shown in Table 1 ).
  • 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.
  • 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- ⁇ ) in the subject during and/or following therapy or by observing a decrease in fibrosis in the subject during and/or following therapy.
  • a change e.g., a decrease
  • one or more inflammatory biomarkers e.g., TGF- ⁇
  • 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.
  • 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
  • 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).
  • a tetracycline may be administered systemically (e.g., orally, or as an injectable) in accordance with standard methods known in the art.
  • 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.
  • 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.
  • 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.
  • suitable skin contact adhesive materials include, but are not limited to, polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates, and polyurethanes.
  • 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.
  • the tetracycline e.g., doxycycline, minocycline, sarecycline, or omadacycline; preferably doxycycline or sarecycline
  • the tetracycline is administered transdermal ⁇ at a submicrobial dose.
  • 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 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.
  • an ointment base may be inert, stable, non-irritating, and non-sensitizing.
  • the tetracycline e.g., doxycycline, minocycline, sarecycline, or omadacycline; preferably doxycycline or sarecycline
  • CNS administration e.g., doxycycline, minocycline, sarecycline, or omadacycline; preferably doxycycline or sarecycline
  • tetracycline 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.
  • carrier molecules such as cationic dendrimers or arginine-rich peptides
  • 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.
  • the tetracycline may be systemically administered (e.g., injected into a vein).
  • 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.
  • the tetracycline e.g., doxycycline, minocycline, sarecycline, or omadacycline; preferably doxycycline or sarecycline
  • the CNS is administered to the CNS at a submicrobial dose.
  • 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.
  • the tetracycline e.g., doxycycline, minocycline, sarecycline, or omadacycline; preferably doxycycline or sarecycline
  • doxycycline or sarecycline is administered orally or rectally at a submicrobial dose.
  • 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
  • tetracyclines for use in treatment of human or animal subjects for inflammatory conditions
  • the tetracycline may be formulated in ways consonant with these parameters.
  • 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.
  • 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, gastrointes
  • 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.
  • 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.
  • 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.
  • 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 may be formulated in a variety of ways that are known in the art.
  • the first and second agents of the combination therapy may be formulated together or separately.
  • the first agent may be a tetracycline
  • the second agent may be an anti-inflammatory agent (e.g., an NSAID).
  • 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.
  • 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.
  • 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,
  • 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).
  • 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
  • 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,
  • 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 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
  • compounds e.g., a tetracycline, such as doxycycline, minocycline, sarecycline,
  • 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.
  • shellac beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl distearate, g
  • 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.
  • 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
  • the liquid forms in which the compounds and compositions of the present invention 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.
  • 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
  • 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.
  • 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
  • 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
  • Tetracyclines for treatment of inflammatory disorders according to the methods and kits of the invention will generally be administered at submicrobial doses.
  • 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.
  • Compounds of the invention may be formulated and employed in combination therapies for the treatment of an inflammatory condition, such as mTBI, OA, or fibromyalgia.
  • 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
  • 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).
  • 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.
  • 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.
  • agents that can be used in a combination therapy with a tetracycline of the invention include, but are not limited to, non-steroidal anti-inflammatory drug (NSAID), a COX-2 inhibitor, capsaicin, and gabapentin.
  • NSAID non-steroidal anti-inflammatory drug
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • kits 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.
  • placebo dosages, or calcium dietary supplements 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- ⁇ , TGF ⁇ 2, or TGF ⁇ 3; preferably TGF- ⁇ ), in a biological sample.
  • a device 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- ⁇ Multispecies ELISA Kit (Life Technologies, Cat. No. KAC1688).
  • Example 1 Treatment of mTBI with doxycycline
  • TGF- ⁇ peripheral circulating levels of TGF- ⁇ will be elevated.
  • NSAIDs and other anti- inflammatory agents have not been largely successful.
  • mitigating the secretion of TGF- ⁇ 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.
  • the study populations are patients with confirmed diagnosis of concussion/ mTBI according to IMPACT criteria.
  • Blood samples will be drawn and biobanked.
  • the output parameters evaluated will include peripheral circulating levels of pro-inflammatory cytokines including TGF- ⁇ 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- ⁇ genes (CC genotype) at the Center for Human Genetics, Inc (Boston). Correlation analyses will be performed between genomic variations of TGF- ⁇ gene expression, patient symptoms and peripheral circulating levels of TGF- ⁇ before and after treatment with
  • TGF- ⁇ 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- ⁇
  • 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 ).
  • an inflammatory biomarker e.g., an inflammatory cytokine, such as those shown in Table 1 .
  • a blood sample may be drawn from the patient and examined for serum levels of TGF- ⁇ . If the level of TGF- ⁇ in the blood sample is found to be elevated relative to a standard, such as the normal level of TGF- ⁇ shown in Table 1 (0-20 ng/ml), or to the level of TGF- ⁇ 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- ⁇ would thus be treated with doxycycline and then monitored for improvement in mTBI symptoms and for normalization of serum TGF- ⁇ levels.
  • an inflammatory biomarker e.g., an inflammatory
  • Diffusion tensor imaging 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.
  • 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.
  • 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).
  • CSF i.e., unrestricted water is free to diffuse in all directions: isotropic
  • 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.
  • 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.
  • 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.
  • 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 Fractional Anisotropy
  • MD Mean Diffusivity
  • 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.
  • the direction of water is equal in all directions (i.e., isotropic), and the value is close to 0.
  • 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 .
  • 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.
  • 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
  • tetracyclines such as doxycycline
  • doxycycline can be used to ameliorate the fibrotic state of the muscle architecture in OA patients.
  • TGF- ⁇ 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- ⁇ can also be assessed during therapy as a measure of therapeutic effectiveness.
  • Genomic DNA will be obtained from peripheral circulating white cells and assayed by polymerase chain reaction for polymorphism of TGF- ⁇ 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- ⁇ gene expression, patient symptoms, quantitation of muscle fibrosis and peripheral circulating levels of TGF- ⁇ 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- ⁇ 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.
  • 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.
  • TGF- ⁇ 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- ⁇ levels will also be measured monthly. We hypothesize that TGF- ⁇ levels will be initially elevated in OA patients, relative to controls lacking OA, and that doxycycline treatment will reduce TGF- ⁇ levels over time.
  • TGF- ⁇ 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- ⁇ , TGF ⁇ 2, or TGF ⁇ 3).
  • an inflammatory biomarker e.g., an inflammatory cytokine
  • Table 1 e.g., TGF- ⁇ , TGF ⁇ 2, or TGF ⁇ 3
  • a blood sample may be drawn from the patient and examined for serum levels of TGF- ⁇ .
  • a patient showing such elevated levels of TGF- ⁇ can then be treated with doxycycline and monitored for improvement in OA symptoms and for normalization of serum TGF- ⁇ 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.
  • Eccentric resistance training of the hamstring has been shown to downregulate TGF- ⁇ .
  • 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.
  • doxycycline will act synergistically with eccentric resistance training, such that patients undergoing both doxycycline treatment and eccentric resistance training show greater downregulation of TGF- ⁇ levels than those undergoing doxycycline treatment or eccentric resistance training alone.
  • Example 11 Treatment of diabetic adhesive capsulitis with doxycycline
  • 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.
  • Patients with fibromyalgia can be treated with doxycycline.
  • 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.
  • 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.
  • Example 14 Treatment of exercise intolerance with doxycycline
  • 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.
  • CPK creatine kinase
  • Example 15 Treatment of hypermobility syndrome with doxycycline
  • the patient was a 17 year old female with hypermobility syndrome.
  • the patient suffered from joint pain involving hands, knees and feet.
  • the patient was placed on a regimen of doxycyline (100 mg per day) for three months.
  • the patient showed complete resolution of all symptoms.
  • the patient 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.
  • JS 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.
  • WBC white blood cell
  • ESR Erythrocyte Sedimentation Rate
  • Example 17 Treatment of mouth ulcers, abdominal pain, and low grade fever with doxycycline
  • 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
  • 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%.
  • VAS visual analog scale
  • Example 19 Treatment of sarcoidosis with hilar adenopathy with doxycycline
  • 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.
  • ANA antinuclear antibodies
  • ACE angiotensis-converting enzyme
  • rheumatoid arthritis and normal muscle enzymes.
  • His ESR was 4

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Abstract

La présente invention concerne des méthodes de traitement de sujets souffrant d'états inflammatoires, tels que lésions traumatiques légères du cerveau (mTBI), arthrose (OA), et fibromyalgie. Lesdites méthodes peuvent consister à déterminer si le sujet présente un tel état inflammatoire et à administrer une quantité efficace d'un composé de tétracycline (par exemple, la doxycycline) audit sujet (par exemple, un humain) L'invention concerne également des procédés permettant de détecter un état inflammatoire chez un sujet en déterminant un niveau d'un biomarqueur inflammatoire, tel que le TGF-βΙ, dans un échantillon biologique (par exemple, du sang) prélevé sur le sujet. De tels procédés peuvent également être utilisés en tant que diagnostic compagnon en combinaison avec un traitement de tétracycline pour évaluer l'efficacité d'un traitement. En outre, l'invention concerne des kits permettant de détecter et de traiter de tels états inflammatoires.
PCT/US2015/024047 2014-04-02 2015-04-02 Méthodes de traitement d'états inflammatoires WO2015153864A2 (fr)

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