Classifications

• • 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
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
  • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
• • 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
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/06—Antipsoriatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

• the present invention relates to methods of treatment for inflammatory disorders, particularly disorders of epithelial tissue such as that of the skin or gastrointestinal tract.
• Leukotriene B 4 is produced by leukocytes, particularly macrophages and monocytes, upon activation by immune complexes, phagocytosis or other stimuli. In this process, membrane phospholipids are broken down by phospholipase A 2 to release arachidonic acid, which is further metabolized via one of two pathways. The first is via cycloxygenases to produce prostaglandins. The second is via lipoxygenases to form leukotriene A 4 ( ⁇ A 4 ). .LTA 4 is converted to LTB or LTC 4 . LTB 4 is a potent chemotactic agent that stimulates neutrophil and macrophage migration (chemotaxis) to sites of inflammation. The structure of LTB 4 is shown below.
• LTB 4 The known pathophysiological responses of LTB 4 include: induction of potent neutrophil chemotactic activity, promotion of adhesion of polymorphonuclear leukocytes (PMN's) to vasculature, increase in vascular permeability, stimulation of the release of lysosomal enzymes, by PMN's.
• PMN's polymorphonuclear leukocytes
• the pro-inflammatory action of LTB 4 has been demonstrated in vivo, wherein topical LTB 4 on human skin promotes the infiltration of PMN's and other inflammatory cells. Intradermal injection of LTB induces accumulation of neutrophils at the injection site, intravenous injection of LTB 4 causes rapid but transient neutropenia (Kingsbury et al, J. Med.
• LTB 4 is believed to interact with two sub-groups of receptor: a high- affinity receptor and a low-affinity receptor. Research indicates that the high- affinity receptor mediates chemotaxis and that the low-affinity receptor mediates LTB 4 -induced secretory and oxidase-activation responses (Yokomizo et al. 2000). Some LTB 4 antagonists are observed to antagonize all LTB 4 mediated activity. Other LTB antagonists modulate only the activity associated with one but not the other sub-population of LTB 4 receptors. - ⁇ -
• LTB 4 Antagonists Compounds, which act as antagonists of LTB 4 include, for example: structural analogs of LTB 4 such as LTB -dimethyl amide and 20-CF 3 -LTB ; SM-9064; U-75302; Ly-223982; SC-41930; ONO 4057 (Prostaglandins, 44(4):261-275, 1992); RG-14893; (E)-3-[2-[6-[3-(3-butoxyphenyl)-3- hydroxypropenyl]pyridin-2-yl]- 1 -hydroxyethyljbenzoate-benzoic acid, lithium salt (Kingsbury J. Med.
• Tofisopam has been shown in humans to have an activity profile that is significantly different from that of widely used 1,4-benzodiazepine (BZ) anxiolytics such as diazepam (Valium®) and chlordiazepepoxide (Librium®).
• BZ 1,4-benzodiazepine
• the 1,4-benzodiazepine in addition to having sedative-hypnotic activity, also possess muscle relaxant and anticonvulsant properties which, though therapeutically useful in some disease states, are nonetheless potentially untoward side effects.
• the 1,4-benzodiazepines though safe when administered alone, may be dangerous in combination with other CNS drugs including alcohol.
• Tofisopam in contrast, is a non-sedative anxiolytic that has no appreciable sedative, muscle relaxant or anticonvulsant properties (Horvath et al., Progress in Neurobiology, 60 (2000), 309-342). In clinical studies, tofisopam improved rather than impaired psychomotor performance and showed no interaction with ethanol (Id.). These observations comport with data that show that tofisopam does not interact with central BZ receptors and binds only weakly to peripheral BZ receptors. Additional studies have shown that tofisopam enhances mitogen-induced lymphocyte proliferation and IL-2 production in vitro.
• GYKI-52466 and GYKI-53655 act as noncompetitive glutamate antagonists at the AMPA ( ⁇ -amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid) site, and have demonstrated neuroprotective, muscle relaxant and anticonvulsant activity (Id.).
• AMPA ⁇ -amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid
• Another group of 2,3- benzodiazepines that have been investigated are represented by the compound GYKI-52895, and show activity as selective dopamine uptake inhibitors with potential use in antidepressant and anti-Parkinsonism therapy.
• Tofisopam (structure shown below), with the atom numbering system indicated) is a racemic mixture of (R)- and (S)- enantiomers. This is due to the asymmetric carbon, i.e., a carbon with four different groups attached, at the 5- position of the benzodiazepine ring.
• tofisopam The molecular structure and conformational properties of tofisopam have been determined by NMR, CD and X-ray crystallography (Visy et al, Chirality 1:271-275 (1989)).
• the 2,3-diazepine ring exists as two different conformers.
• the 5-ethyl group is positioned quasi-axially in the minor conformers, (-)R and (+)S.
• racemic tofisopam can exist as four molecular species, i.e., two enantiomers, each of which exists as two conformations.
• IBD Inflammatory Bowel Disease Crohn's disease
• UC ulcerative colitis
• IBD indeterminate colitis and infectious colitis
• mflammatory bowel diseases are chronic recurrent inflammatory diseases of unclear etiology, affecting the small intestine and colon. IBD can involve either or both the small and large bowel. These disorders fall into the category of "idiopathic" IBD because the etiology for them is unknown. Pathologic findings are generally not specific, although they may suggest a particular form of IBD.
• Active IBD is characterized by acute inflammation.
• Chronic IBD is characterized by architectural changes of crypt distortion and scarring.
• crypt refers to a deep pit that protrudes down into the connective tissue surrounding the small intestine. Crypt abscesses (active IBD characterized by the presence of neutrophils in crypt lumens) can occur in many forms of IBD, not just UC. Under normal conditions the epithelium at the base of the crypt is the site of stem cell proliferation and the differentiated cells move upwards and are shed 3-5 days later at the tips of the villi. This normal process, necessary for proper bowel function, is interrupted by IBD. UC involves the colon as a diffuse mucosal disease with distal predominance. The rectum is virtually always involved, and additional portions of colon may be involved extending proximally from the rectum in a continuous pattern.
• CD Crohn's disease
• UC ulcerative colitis
• ileocolitis a chronic inflammatory disease that has periods of remission (time when person feels well) and relapse (when a person feels ill).
• CD is an inflammation and ulceration process that occurs in the deep layers of the intestinal wall. The most common areas affected are the lower part of the small intestine, called the ileum, and the first part of the colon. This type of CD is called ileocolitis.
• CD can infrequently affect any part of the upper gastrointestinal tract. Aphthous ulcers, which are similar to cold sores, are common.
• Ulcers can also occur in the esophagus, stomach and duodenum.
• Therapy for IBD has historically included administration of corticosteroids.
• drawbacks of long term corticosteroid therapy include masking (or induction) of intestinal perforation, osteonecrosis and metabolic bone disease. Additional problems relate to development of corticosteroid dependency (Habnauer, New England Journal of Medicine, 334(13), p 841-848, 1996).
• Aminosalicylates such as sulfasalazine and mesalamine have been used to treat mild or moderately active UC and CD, and to maintain remission (Id at 843).
• Immunomodulatory drugs such as azathioprine and mercaptopurine have been used in long term treatment for patients with IBD. Common complications with both of these drugs include pancreatitis, which occurs with an incidence of 3-15% of patients, and bone marrow suppression, which requires regular monitoring. More potent immunosuppressive drugs such as cyclosporine and methotrexate have been employed, but toxicity of these drugs limits their use to specific situations of refractory disease states. Other therapeutic approaches include antibiotic therapy and nutritional therapy. Often, therapy involves a combination of the above-described drug therapies in addition to surgical resection of the bowel. There is no cure for IBD.
• IBD chronic and progressive nature of IBD demands a long-term treatment that maximizes the local antiinflammatory effect while minimizing the global systemic effect on the immune system.
• Chronic inflammatory disorders such as CD typically demonstrate periods of remission between intervals when the inflammatory is active and requires acute treatment. This is an example of a circumstance wherein it is known beforehand that an individual will develop, or is likely to develop an inflammatory disorder.
• Irritable bowel syndrome IBS is a disorder of the bowel which is distinct from IBD. IBS affects at least 10% to 20% of adults in the U.S. IBS is the most common disorder diagnosed by gastroenterologists and one of the top ten most frequently diagnosed conditions among U.S. physicians.
• IBS is classified as a "functional gastrointestinal disorder," which means there is a disturbance in bowel function.
• IBS is not a considered a disease, but rather a syndrome, i.e., a group of symptoms.
• the symptoms typically include chronic abdominal pain/discomfort, and irregular bowel function, e.g., diarrhea, constipation, or alternating diarrhea and constipation.
• IBS does not cause inflammation.
• IBS sufferers show no sign of disease or abnormalities on examination of the colon.
• IBD and IBS share some similar symptoms, particularly cramping and diarrhea, the underlying disease process is quite different. IBD involves inflammation or destruction of the bowel wall, which can lead to deep ulcerations and narrowing of the intestines.
• IBS is a disorder of the gastrointestinal (GI) tract for which no apparent cause can be found.
• An individual can simultaneously have both IBS and an inflammatory disorder such as IBD. When this occurs, imprecise diagnosis may lead to inadequate medical intervention.
• IBS Inflammatory Skin Disorders: 1. Psoriasis. Another chronic inflammatory condition believed to be mediated by
• LTB 4 is psoriasis.
• Psoriasis is a chronic, recurrent, papulosquamous plaque on areas of trauma such as the elbow, knee or scalp, though it may appear elsewhere on the skin. Psoriasis may coexist with lupus er ⁇ thematosis in some individuals. Current treatments include topical administration of psoralens. "Psoralens” refers to a group of substances found in many different plants, especially psoralea corylifolia. Psoralens interact with nucleic acids and are also used as research tools. Psoriasis is also treated by long-wave ultraviolet radiation. Neither treatment cures or prevents recurrence of psoriasis symptoms. 2.
• Atopic Dermatitis/Eczema Atopic Dermatitis/Eczema.
• Atopic dermatitis is a chronic disease that affects the skin. In atopic dermatitis, the skin becomes extremely itchy. Scratching leads to redness, swelling, cracking, "weeping" clear fluid, and finally, crusting and scaling. In most cases, there are periods of exacerbations followed by periods of remissions. Although it is difficult to identify exactly how many people are affected by atopic dermatitis, an estimated 20% of infants and young children experience symptoms of the disease. Approximately 60% of these infants continue to have one or more symptoms of atopic dermatitis in adulthood. Thus, more than 15 million people in the United States have symptoms of the disease 3. Contact Dermatitis.
• RA Rheumatoid Arthritis
• Another chronic inflammatory disorder believed to be mediated by LTB 4 is RA, which is an autoimmune disease of the joints.
• RA is characterized by the following criteria 1-7, wherein criteria 1-4 are present for more than 6 weeks: (1) morning stiffness in and around joints lasting at least one hour before maximum improvement; (2) soft tissue swelling (arthritis) of three or more joints observed by a physician; (3) swelling (arthritis) of the proximal interphalangeal, metacarpal phalangeal, or wrist joints; (4) symmetric swelling; (5) rheumatoid nodules, i.e., a granulomatous lesion characterized by central necrosis encircled by a palisade of monocytes and an exterior mantle of lymphocytic infiltrate.
• criteria 1-7 wherein criteria 1-4 are present for more than 6 weeks: (1) morning stiffness in and around joints lasting at least one hour before maximum improvement; (2) soft tissue swelling (arthritis) of three or more joints observed by a physician; (3) swelling (arthritis) of the proximal interphalangeal, metacarpal phalangeal, or wrist joints; (4)
• RA is a chronic disorder for which there is no known cure.
• the major goals of treatment of RA are to reduce pain and discomfort, prevent deformities and loss of joint function, and maintain a productive and active life. Inflammation must be suppressed and mechanical and structural abnormalities corrected or compensated by assistive devices. Treatment options include reduction of joint stress, physical and occupational therapy, drug therapy, and surgical intervention.
• NSAID's non-steroidal anti-inflammatory agents
• corticosteroids corticosteroids
• DMARD's remittive agents or disease modifying anti-rheumatic drugs
• DMARD's include leflunomide (AravaTM), etanercept (EnbrelTM), infliximab (RemicadeTM), antimalarials, methotrexate, gold salts, sulfasalazine, d- penicillamine, cyclosporin A, cyclophosphamide and azathioprine. Because cartilage damage and bony erosions frequently occur within the first two years, rheumatologists now move more aggressively to a DMARD agent. Treatment of RA by chronic administration of a corticosteroid involves the same side effect profile as discussed regarding IBD above. Chronic administration of NSAID's also produces side effects. The most common toxicity of NSAID's is gastrointestinal disturbance.
• Gout is another inflammatory disorder believed to be mediated by LTB 4 .
• Gout is characterized by a disturbance of uric-acid metabolism occurring chiefly in males.
• Gout is characterized by painful inflammation of the joints, especially of the feet and hands, and arthritic attacks resulting from elevated levels of uric acid in the blood and the deposition of urate crystals around the joints. The condition can become chronic and result in deformity.
• Gout can present another circumstance wherein it is known beforehand that an individual will or is likely to develop an inflammatory disorder. In the instance of patients undergoing radiotherapy or chemotherapy, the individual may experience a dramatic rise in serum uric acid levels associated with lysis of the tumor mass.
• prophylaxis with an LTB 4 antagonist can act to prevent the inflammatory condition of gout.
• Radiation-induced Gastrointestinal Inflammation Radiation-induced Gastrointestinal Inflammation. Radiation-induced gastrointestinal inflammation is another inflammatory disorder believed to be mediated by LTB 4 . Radiation works by damaging cancer cells, but unfortunately can damage non-diseased tissue as well, causing a typical inflammatory reaction in response. Therapeutic radiation is thus generally applied to a defined area of the subject's body which contains abnormal proliferative tissue in order to maximize the dose absorbed by the abnormal tissue and minimize the dose absorbed by the nearby normal tissue.
• Mucositis involves ulcerative breakdown of mucosal epithelial tissue, and is literally defined as inflammation of the mucous membrane.
• Mucositis may be characterized by the following phases: 1. Early inflammatory phase characterized by release of inflammatory cytokines in response to local tissue damage caused by cytotoxic agent(s); 2. Epithelial phase characterized by death of basal cells, which hinders re-population of the epithelium. This inability to regenerate leads to atrophy followed by ulceration.
• the ulceration represents loss of an important anatomic barrier at a site of local microflora; 3. Infection phase characterized by local invasion of microflora that results in an inflammatory response to the local infection. The inflammation results in additional local tissue damage and possibly erosive ulceration; and 4. Hair phase characterized by resolution of the infection and regeneration of epithelium.
• Oral mucositis produces the following clinical symptoms and signs resulting from cellular damage: 1) sensation of dryness; 2) asymptomatic redness and erythema; 3) solitary white elevated desquamative patches which are painful upon pressure contact; and 4) large, painful, contiguous pseudomembranous lesions associated with dysphagia and decreased oral intake.
• gastrointestinal mucosal damage results from disturbance of cellular mitosis that leads to reduction in the turnover rate of the basal cells of the intestinal crypts.
• the symptoms and signs of gastrointestinal mucositis include tenesmus (painful ineffectual straining at stool), pain, bleeding, diarrhea, telangectasia (neovascularization), and progression to ulceration.
• Early signs of diarrhea include increased stool frequency, loose or watery stool, food aversion, increased bowel sounds, abdominal pain, and some loss of skin turgor indicative of dehydration.
• Necrotizing enterocolitis is an inflammatory disease of unknown etiology that afflicts between 1-5% of all infants admitted to neonatal intensive care units, most of whom are premature infants. Signs and symptoms include abdominal distention, gastrointestinal hemorrhage, and feeding intolerance. The disease most often involves the ileum and colon, and is characterized by loss of epithelium and submucosal edema, ulcerations, and, in severe cases, transmural necrosis.
• H. Aphthous Ulcers (Oral). Although the cause of aphthous ulcers remains unknown, many physicians believe they are caused by autoimmune phenomena, which cause the destruction of discrete areas of the oral mucosa which leads to oral ulceration. Among the cytokines present in these active areas of ulceration, TNF- ⁇ appears to play a predominant role.
• I. Gingivitis/Periodontitis Adult periodontitis is strongly associated with infection by Porphyromonas gingivalis. Proteolytic enzymes, which are produced in large quantity by this bacteria, are considered as important pathogenic agents.
• GCF gingival crevicular fluid
• Pharyngitis Pharyngitis is defined as an infection or irritation of the pharynx and/or tonsils. The etiology is usually infectious, with 40-60% of cases being of viral origin and 5-40% of cases being of bacterial origin. Other causes include allergy, trauma, toxins, and neoplasia.
• Conjunctivitis is an inflammation of the conjunctivae, which are the mucous membranes covering the white of the eyes and the inner side of the eyelids.
• Bacterial conjunctivitis is an infection caused by bacteria.
• Viral conjunctivitis may be caused by a virus called 'adenovirus'.
• Chlamydial conjunctivitis is caused by an organism called Chlamydia trachomatis.
• Allergic conjunctivitis is common in people who have other signs of allergic disease such as hay fever, asthma and eczema.
• Reactive conjunctivitis (chemical or irritant conjunctivitis) is caused by a chemical irritant, e.g., chemicals in swimming pools, smoke or solvent fumes.
• M. Peptic Ulcer Disease Inhibition of gastric acid secretion with H 2 -receptor antagonists and, more recently, blockers of H + , K + -ATPase (also known as the proton pump) has been the mainstay of therapy for peptic ulcer disease. The pathophysiology of peptic ulcers remains obscure.
• H. pylori has been widely accepted as the cause of chronic, active, type B gastritis. Further, this form of gastritis has been linked directly to peptic ulcer disease by studies showing that eradication of H. pylori reverses this gastritis and prevents duodenal ulcer relapse.
• Particular classes of compounds which have been investigated include aminosalicylates, corticosteroids, antimetabolites, immunosuppressants, tumor necrosis factor alpha (TNF- ⁇ ) inhibitors, inhibitors of leukotriene synthesis e.g., 5-lipoxygenase (5-LO) inhibitors, and leukotriene antagonists.
• TNF- ⁇ tumor necrosis factor alpha
• inhibitors of leukotriene synthesis e.g., 5-lipoxygenase (5-LO) inhibitors
• leukotriene antagonists Exemplary compounds of interest that have been shown to possess activity in models of inflammatory disorders are listed in Table 1 along with putative mechanisms of action and the indications for which they have been investigated.
• New antiinflammatory agents are needed which are useful in the treatment of inflammatory disorders such as IBD, RA, gout, psoriasis and radiation-induced gastrointestinal inflammation.
• agents are needed that are appropriate for chronic long-term use in treatment.
• agents are needed that are useful in the prevention of inflammatory disorders, particularly LTB 4 -mediated inflammatory disorders that occur secondary to observable events such as ionizing radiation therapy.
• Summary of the Invention in one embodiment of the invention there is provided a method of treatment or prevention of inflammatory disorders in an individual, particularly, inflammatory disorders affecting epithelial tissues, in particular, IBD, including CD and, UC, psoriasis; rheumatoid arthritis; gout and radiation-induced gastrointestinal inflammation.
• the method comprises administering to the individual an effective amount of at least one compound according to formula I:
• R 1 is -(C 1 -C 7 )hydrocarbyl or -(C 2 -C 6 )heteroalkyl;
• R is selected from the group consisting of -H, and -(d-C ⁇ hydro- 1 9 carbyl, wherein R and R may combine to form a carbocyclic or heterocyclic 5- or 6-membered ring;
• R 3 is independently selected from the group consisting of -O( - C 6 )alkyl, -OH, -O-acyl, -SH, -S(C 1 -C 3 )alkyl, -NH 2 , -NH(C 1 -C 6 )alkyl, -N((C C 6 )alkyl) 2 , -NH-acyl, -NO 2 and halogen;
• n is 1, 2 or 3;
• R 4 and R 5 are independently selected from the group consisting of -O(C C 6 )alkyl, -OH,
• the compound is administered at a dose of less than about 25 mg/day, more preferably less than aboutlO mg/day and even more preferably at a dose of less than about 1 mg/day.
• Preferred liquid formulations are administered at a dose of less than about 10 mg/ml and more preferably at a dose of less than about 1 mg/ml.
• the administered compounds according to formula I comprise a racemic mixture of compounds with respect to the absolute conformation at the 5-position of the benzodiazepine ring.
• the administered compounds according to formula I comprise an (i?)-enantiomer, substantially free of the corresponding (S)-enantiomer of the same compound, with respect to the absolute conformation at the 5-position of the benzodiazepine ring,
• the administered compound of formula I comprising an (R)- enantiomer, or a pharmaceutically-acceptable salt of such a compound comprises 85% or more by weight of the (i?)-enantiomer.
• the administered compound of formula I comprising an (i?)-enantiomer, or a pharmaceutically-acceptable salt of such a compound comprises 90% or more by weight of the (i?)-enantiomer. Even more preferably, the administered compound of formula I comprising an (i?)-enantiomer, or a pharmaceutically- acceptable salt of such a compound, comprises 95% or more by weight of the (i?)-enantiomer. Most preferably, the administered compound, comprising an (i?)-enantiomer of formula I, or a pharmaceutically-acceptable salt of such a compound, comprises 99% or more by weight of the (u)-enantiomer.
• R 1 is -(C ⁇ -C 6 )al yl;
• R 2 is selected from the group consisting of-H and -(Ci-C 6 )alkyl;
• R 3 is independently selected from the group consisting of -O(Ci- C 6 )alkyl, -O-acyl and -OH;
• n is 1, 2 or 3;
• R 4 and R 5 are independently selected from -O(C 1 -C 6 )alkyl, -O-acyl and -OH, wherein, R 4 and R 5 may combine to form a 5, 6 or 7-membered heterocyclic ring; or a pharmaceutically-acceptable salt of such a compound.
• R 1 is -CH 2 CH 3 ;
• R 2 is -(C ⁇ -C 6 )alkyl;
• R 3 , R 4 and R 5 are independently selected from the group consisting of
• R 1 is -CH 2 CH 3 ;
• R 2 is -CH 3 ;
• R 3 , R 4 and R 5 are independently selected from the group consisting of -OH and -OCH 3 ; and
• n is 1, 2 or 3; or a pharmaceutically-acceptable salt of such a compound.
• R 2 is -CH 3 ; > R 3 , R 4 and R 5 are independently selected from the group consisting of -OH and -OCH 3 ; and n is 2 ; or a pharmaceutically-acceptable salt of such a compound.
• R 1 is -CH 2 CH 3 ;
• R 2 is -CH 3 ;
• R 3 , R 4 and R 5 are independently selected from the group consisting of -OH and -OCH 3 ;
• n is 2; and wherein R 3 comprises substituents at the 3- and 4- positions of the phenyl ring; or a pharmaceutically-acceptable salt of such a compound.
• Preferred racemic compounds according to formula I, for administration are selected from the group consisting of: l-(3,4-dimethoxyphenyl)-4-methyl-5-ethyl-7,8-dimethoxy-5H-2,3- benzodiazepine; l-(3,4-dimethoxyphenyl)-4-methyl-5-ethyl-7-hydroxy-8-methoxy-5H- 2,3-benzodiazepine; l-(3-hydroxy-4-methoxyphenyl)-4-methyl-5-ethyl-7,8-dimethoxy-5H- 2,3-benzodiazepine; l-(3-methoxy-4-hydroxyphenyl)-4-methyl-5-ethyl-7,8-dimethoxy-5H-
• 2,3-benzodiazepine l-(3,4-dimethoxyphenyl)-4-methyl-5-ethyl-7-methoxy-8-hydroxy-5H- 2,3-benzodiazepine; l-(3-methoxy-4-hydroxyphenyl)-4-methyl-5-ethyl-7-hydroxy-8- methoxy-5H-2,3-benzodiazepine; 1 -(3-hydroxy-4-methoxyphenyl)-4-methyl-5-ethyl-7-hydroxy-8- methoxy-5H-2,3-benzodiazepine; and pharmaceutically-acceptable salts of such compounds.
• the compound according to formula I, for administration is l-(3,4-dimethoxyphenyl)-4-methyl-5-ethyl-7,8-dimethoxy-5H- 2,3-benzodiazepine; or a pharmaceutically-acceptable salt thereof.
• Preferred compounds comprising (i?)-enantiomers according to formula I, for administration are selected from the group consisting of: (i?)-l-(3,4-dimethoxyphenyl)-4-methyl-5-ethyl-7,8-dimethoxy-5H-2,3- benzodiazepine; (i?)-l-(3,4-dimethoxyphenyl)-4-methyl-5-ethyl-7-hydroxy-8-methoxy- 5H-2,3-benzodiazepine; (i?)-l-(3-hydroxy-4-methoxyphenyl)-4-methyl-5-ethyl-7,8-dimethoxy- 5H-2,3-benzodiazepine; (i?)-l-(3-methoxy-4-hydroxyphenyl)-4-methyl-5-ethyl-7,8-dimethoxy- 5H-2,3-benzodiazepine; (i?)-l-(3-methoxy-4-hydroxyphenyl)-4-methyl-5-ethyl-7,8
• the compound according to formula I, for administration is (i?)-l-(3,4-dimethoxyphenyl)-4-methyl-5-ethyl-7,8-dimethoxy- 5H-2,3-benzodiazepine, substantially free of the corresponding (S)-enantiomer; or a pharmaceutically-acceptable salt thereof.
• the compound, (i?)-l-(3,4-dimethoxyphenyl)-4-methyl-5-ethyl-7,8- dimethoxy-5H-2,3 -benzodiazepine, the (i?)-enantiomer of tofisopam, is shown in the structure diagram below.
• the aforesaid compounds are used in the preparation of medicaments for treating or preventing an inflammatory disorder.
• a method of treatment or prevention of inflammatory disorders in an individual comprising administering to the individual an effective amount of at least one compound according to formula I as defined herein in combination with at least one additional therapeutic agent.
• the additional therapeutic agent is selected, from the group consisting of aminosalicylates, corticosteroids, antimetabolites, immunosuppressants, TNF- ⁇ inhibitors, 5-LO inhibitors and leukotriene antagonists, wherein the leukotriene antagonist is not a compound of formula I.
• the invention also encompasses a method of addressing one or a combination of inflammatory disorders of epithelial tissue in a mammal in which an unexpectedly low amount of the compound can be used. More specifically, it has been found that it is possible to achieve good anti- inflammatory activity when the compound is contacted directly on the skin (i.e., adminstered topically).
• Preferred administration routes generally include contacting the epithelium with a formulation in which the compound is present in an amount well below about 100 mg/ml, for example, about 50 mg/ml or well below 10 mg/ml, for example 1 mg/ml or O.lmg/ml.
• a preferred formulation is a topical formulation.
• the compounds of Formula I are particularly useful to address one or a combination of gastrointestinal disorders in a mammal such as a human patient.
• the invention features a method for preventing, treating, reducing the severity of, or delaying onset of a gastrointestinal disorder that includes administering to the patient an effective amount of the compound.
• the compound can be used alone as the sole active agent or in combination with one, a few or several other agents including other compounds according to Formula I.
• Preferred administration routes include those generally well suited for gastrointestinal delivery such as intracolonic delivery (eg an enema or suppository) and other delivery methods described below. Definitions
• the term "inflammation" or inflammatory response" refers to a defense reaction of living tissue to injury.
• the response serves to contain and to repair the injury. Multiple chemical mediators of inflammation derived from either plasma or cells have been observed. Compounds produced in the metabolism of arachidonic acid, such as prostaglandins and leukotrienes, also affect inflammation, leukotrienes mediating essentially every aspect of acute inflammation.
• An "inflammatory disorder mediated by LTB " or a "LTB 4 -mediated disorder” refers to a disorder resulting from an inflammatory response wherein LTB 4 mediation is implicated as a factor by observation of LTB 4 presence at the site of the inflammation.
• An "inflammatory disorder of an epithelial tissue” or “of an epithelium” refers to an inflammatory disorder in which one or more epithelial tissues or tissues adjacent to the epithelial layer are affected.
• Exemplary epithelial tissues include the epidermal layer of the skin, the cornea epithelium of the eye, and the epithelia associated with the mucosal linings of the respiratory, alimentary, gastrointestinal and urinary tracts.
• the term "receptor” refers to a molecular structure or site on the surface or interior of a cell that binds with substances such as hormones, antigens, drugs, or neurotransmitters.
• An "agonist" at a receptor refers to a drug or other chemical that can bind to a receptor to produce the physiologic reaction that is typical of a naturally occurring substance.
• an "antagonist” refers to a chemical substance that acts at a receptor to produce a physiologic reaction that is other than the action produced by the natural, endogenous receptor-binding entity or natural ligand. Such antagonist activity may occur when a drug or chemical substance binds the receptor at a much lower concentration than the natural ligand, and thereby displaces the natural ligand and prevents or reduces the amount of receptor binding to the natural ligand.
• LTB 4 antagonist means a chemical substance that competitively binds to the LTB 4 receptor such that (a) the binding of the natural ligand (LTB 4 ) is inhibited by occupation of the LTB 4 receptor by the LTB 4 antagonist, and (b) the LTB 4 antagonist bound to the LTB 4 receptor does not generate the same physiological response produced by native LTB 4 bound to the LTB 4 receptor.
• the alkyl portion is preferably (d-C 6 )alkyl, more preferably (C 1 -C 3 )alkyl.
• the R is hydrocarbyl, it is preferably (d- C 7 )hydrocarbyl.
• R is hydrocarbyl, it is preferably alkyl, more preferably (d-C 6 )alkyl.
• alkyl by itself or as part of another substituent means, unless otherwise stated, a straight, branched or cyclic chain hydrocarbon radical, including di- and multi-radicals, having the number of carbon atoms designated (i.e. Ci-C ⁇ means one to six carbons).
• Alkyl groups include straight chain, branched chain or cyclic groups, with straight being preferred. Examples include: methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl, cyclohexyl and cyclopropylmethyl.
• (d-C 6 )alkyl is preferred. Most preferred is (d-C 3 )alkyl, particularly ethyl, methyl and isopropyl.
• alkoxy employed alone or in combination with other terms means, unless otherwise stated, an alkyl group having the designated number of carbon atoms, as defined above, connected to the rest of the molecule via an oxygen atom, such as, for example, methoxy, ethoxy, 1-propoxy, 2-propoxy (isopropoxy) and the higher homologs and isomers.
• oxygen atom such as, for example, methoxy, ethoxy, 1-propoxy, 2-propoxy (isopropoxy) and the higher homologs and isomers.
• Preferred are (d- C 6 )alkoxy. More preferred is (d-C 3 )alkoxy, particularly ethoxy and methoxy.
• amine or “amino” refers to radicals of the general formula
• R and R' are independently selected from hydrogen or a hydrocarbyl radical, or wherein R and R' combined form a heterocycle.
• amino groups include: -NH 2 , methyl amino, diethyl amino, anilino, benzyl amino, piperidinyl, piperazinyl and indolinyl.
• Preferred hydrocarbyl radicals are (d-C ⁇ hydrocarbyl radicals.
• Preferred are hydrocarbyl radicals that are alkyl radicals. More preferred are (d-C 6 )alkyl.
• aromatic refers to a carbocycle or heterocycle having one or more polyunsaturated rings having aromatic character (4n + 2) delocalized ⁇ (pi) electrons).
• aryl employed alone or in combination with other terms, means, unless otherwise stated, a carbocyclic aromatic system containing one or more rings (typically one, two or three rings) wherein such rings may be attached together in a pendent manner, such as a biphenyl, or may be fused, such as naphthalene. Examples include phenyl; anthracyl; and naphthyl.
• hydrocarbyl refers to any moiety comprising only hydrogen and carbon atoms. This definition includes for example alkyl, alkenyl, alkynyl, aryl and benzyl groups. Preferred are (C 1 -C )hydrocarbyl.
• heteroalkyl by itself or in combination with another term means, unless otherwise stated, a stable straight or branched chain radical consisting of the stated number of carbon atoms and one or two heteroatoms selected from the group consisting of O, N, and S. Nitrogen and sulfur atoms may be optionally oxidized to the N-oxide and sulfoxide or sulfone, respectively. In addition, a nitrogen heteroatom may be optionally quaternized. The heteroatom(s) may be placed at any position of the heteroalkyl group, including between the rest of the heteroalkyl group and the fragment to which it is attached, as well as attached to the most distal carbon atom in the heteroalkyl group.
• heteroatoms may be consecutive, such as, for example, -CH 2 -NH-OCH 3 , or -CH 2 -CH 2 -S-S-CH 3 . More preferred are heteroalkyl groups containing one or two oxygen atoms. When two groups may "combine to form a carbocyclic or heterocyclic 5- or 6-membered ring," a carbocyclic ring is preferably saturated. Preferred heterocyclic rings are saturated rings containing one or two heteroatoms selected ( from N, O and S.
• Heterocyclic rings annulated to the benzodiazepine seven- membered ring in this way include, for example, furan, dihydrofuran, tetrahydrofuran, pyran, dihydropyran, tetrahydropyran, thiophene, dihydrothiophene, tefrahydrothiophene, pyrrole, dihydropyrrole, pyrrolidine, pyridine, dihydropyridine, tetrahydropyridine and piperidine.
• preferred heterocyclic rings are 5- or 6-membered rings containing one or two heteroatoms selected from N, O and S.
• heterocyclic rings containing one heteroatom selected from N, O and S More preferred are heterocyclic rings containing one heteroatom selected from N, O and S.
• Heterocyclic rings annulated to the benzodiazepine phenyl ring in this way include, for example, furan, dihydrofuran, dioxane, dioxolane, pyran, dihydropyran, tetrahydropyran, thiophene, dihydrothiophene, pyridine, dihydropyridine, tetrahydropyridine, piperidine, pyrrole, dihydropyrrole, imidazole, dihydroimidazole, thiazole, dihydrothiazole, oxazole, and dihydrooxazole.
• substituted means that an atom or group of atoms has replaced hydrogen as the substituent attached to another group.
• substituted refers to any level of substitution, namely mono-, di-, hi-, tetra-, or penta-substitution, where such substitution is permitted.
• the substituents are independently selected, and substitution may be at any chemically accessible position.
• optically active refers to a property whereby a material rotates the plane of plane-polarized light. A compound that is optically active is nonsuperimposable on its mirror image. The property of nonsuperimposablity of an object on its mirror image is called chirality.
• the property of "chirality" in a molecule may arise from any structural feature that makes the molecule nonsuperimposable on its mirror image.
• the most common structural feature producing chirality is an asymmetric carbon atom, i.e., a carbon atom having four nonequivalent groups attached thereto.
• the term "enantiomer” refers to each of the two nonsuperimposable isomers of a pure compound that is optically active. Single enantiomers are designated according to the Cahn-Ingold-Prelog system, a set of priority rules that rank the four groups attached to an asymmetric carbon. See March, fh
• the molecule is oriented so that the lowest ranking group is pointed away from the viewer. Then, if the descending rank order of the other groups proceeds clockwise, the molecule is designated R and if the descending rank of the other groups proceeds counterclockwise, the molecule is designated S.
• R the descending rank order of the other groups proceeds clockwise
• S the descending rank of the other groups proceeds counterclockwise
• the molecule is designated S.
• Racemate or the phrase “racemic mixture” refers to a 50-50 mixture of the (R)- and (S)-enantiomers of a compound such that the mixture does not rotate plane-polarized light.).
• substantially isolated or “substantially free” of the other enantiomer or the term “resolved” or the phrase “substantially free” of the corresponding (S)-enantiomer, when used to refer to an optically active compound of formula I, means the (/.)- and (S)-enantiomers of the compound have been separated such that the composition is 80% or more by weight a single enantiomer.
• (i?)-2,3-benzodiazepine substantially free of the (S)- enantiomer is meant a 2,3-benzodiazepine compound that comprises 80% or more by weight of its (i?)-enantiomer and likewise contains 20% or less of its (S)-enantiomer as a contaminant, by weight.
• an inflammatory disorder particularly a LTB 4 - mediated inflammatory disorder
• an inflammatory disorder refers to the amount of a compound of formula I, or of a combination of a compound of formula I with one or more additional agents, e.g., aminosalicylates, corticosteroids, antimetabolites, immunosuppressants, TNF- ⁇ inhibitors, inhibitors of leukotriene synthesis, or leukotriene antagonists, that inhibits the inflammatory process.
• additional agents e.g., aminosalicylates, corticosteroids, antimetabolites, immunosuppressants, TNF- ⁇ inhibitors, inhibitors of leukotriene synthesis, or leukotriene antagonists.
• the inhibition of the inflammatory process results in a therapeutically useful and selective reduction in the symptoms of inflammation when administered to a patient suffering from a disorder which manifests chronic or acute inflammation, particularly inflammation associated with physiologically relevant concentrations of LTB 4 .
• An effective amount of a compound of formula I, or of a combination of a compound of formula I with one or more additional agents, e.g., aminosalicylates, corticosteroids, antimetabolites, immunosuppressants, TNF- ⁇ inhibitors, inhibitors of leukotriene synthesis, or leukotriene antagonists, for the prevention of an inflammatory disorder, particularly a LTB -mediated inflammatory disorder, is an amount which prevents or delays the onset of symptoms of an inflammatory disorder in an individual during a time interval coinciding with an increased risk of the inflammatory disorder.
• an inflammatory disorders particularly LTB -mediated inflammatory disorders
• these terms refer, unless the context indicates otherwise, to an organism that is afflicted with such an inflammatory disorder.
• preventing inflammatory disorders particularly, preventing LTB -mediated inflammatory disorders
• this term refers unless the context indicates otherwise, to an organism that is likely to be afflicted with such an inflammatory disorder.
• the selection of an individual likely to incur such an inflammatory disorder may take into account the presence of inflammatory conditions that historically are known to have a high incidence of recurrence, such as, for example, IBD.
• the likelihood of incurring such an inflammatory disorder may also be due to tissue insult that is known beforehand, such as a surgical procedure.
• the future inflammatory disorder may also result from a secondary effect of an initial tissue insult.
• An example of this is inflammation due to gout caused by elevated uric acid levels that occur secondary to lysis of a tumor mass following administration of cytotoxic chemotherapy or therapeutic radiation treatment.
• prevention in this context also includes a delay in the onset of inflammation or the symptoms thereof or a prolongation of periods of remission in an individual who experiences recurring inflammatory disorders.
• Fig. 1 is a plot of data gathered in a competitive binding study of the displacement of [ 3 H]LTB 4 by (i?)-tofisopam from LTB 4 receptors. IC 50 and K ⁇ values for (i?)-tofisopam displacement of [ 3 H]LTB 4 were generated.
• Fig. 2 is a plot of data gathered in a competitive binding study of the displacement of [ 3 H]LTB by racemic tofisopam from LTB receptors. IC 50 and K; values for racemic tofisopam displacement of [ 3 H]LTB 4 were generated.
• Fig. 1 is a plot of data gathered in a competitive binding study of the displacement of [ 3 H]LTB 4 by (i?)-tofisopam from LTB 4 receptors. IC 50 and K ⁇ values for (i?)-tofisopam displacement of [ 3 H]LTB 4 were generated.
• Fig. 2 is a plot of data gathered in a competitive binding study of the displacement
• FIG. 3 is a plot of data gathered in a competitive binding study of the displacement of [ 3 H]LTB 4 by (S)-tofisopam from LTB receptors. IC 50 and Ki values for (S)-tofisopam displacement of [ H]LTB 4 were generated.
• 2,3-benzodiazepines of formula I are antagonists of leukotriene B 4 .
• Inflammatory disorders believed to be treatable or preventable by methods of the invention include, for example: inflammatory bowel diseases (e.g., CD, UC, indeterminate colitis, and infectious colitis); RA; gout; mucositis (e.g., oral mucositis, gastrointestinal mucositis, nasal mucositis, and proctitis); necrotizing enterocolitis; inflammatory skin disorders (e.g., psoriasis, atopic dermatitis, and contact hypersensitivity); aphthous ulcers; pharyngitis; esophagitis; peptic ulcers; gingivitis; periodontitis; ocular diseases (e.g., conjunctivitis, retinitis, and uveitis); and radiation-induced gastrointestinal inflammation.
• inflammatory bowel diseases e.g., CD, UC, indeterminate colitis, and infectious colitis
• RA e.g., inflammatory bowel diseases
• compounds of formula I are believed useful in the treatment of the above inflammatory disorders in an individual who is also suffering from IBS.
• Preparation of 2,3-Benzodiazepines of Formula I The (i?)-2,3-benzodiazepines of formula I useful in the present invention may be prepared by one of several methods. These methods generally follow the synthetic strategies and procedures used in the synthesis of racemic 2,3- benzodiazepines of formula I, such as tofisopam and tofisopam analogs. See
• an “(i?)-2,3 -benzodiazepine” is meant a 2,3 -benzodiazepine that has an (R) absolute conformation by virtue of a substitution at the 5-position of the benzodiazepine ring to give a resolvable chiral carbon at the 5-position.
• an “(i?)-2,3- benzodiazepine substantially free of the (S)-enantiomer” or "an (i?)-enantiomer of a compound of formula I substantially free of the corresponding (S)- enantiomer” is meant a compound that comprises 80% or more by weight of the desired (R)-enantiomer and likewise contains 20% or less of the (S)-enantiomer as a contaminant, by weight.
• compounds used in methods of the present invention have a composition that is 85% by weight or greater of the (i?)-enantiomer, and 15% by weight, or less, of the (S)-enantiomer. More preferably, compounds used in methods of the present invention have a composition that is 90% by weight or greater of the (i?)-enantiomer and 10% by weight, or less, of the (S)-enantiomer. More preferably, compounds used in methods of the present invention have a composition that is 95% by weight or greater of the (/ ⁇ -enantiomer and 5% by weight, or less, of the (S)-enantiomer.
• compounds used in methods of the present invention have a composition that is 99% by weight or greater of the (i?)-enantiomer and 1% by weight, or less, of the (S)-enantiomer.
• Racemic 2,3-benzodiazepines of formula I may be synthesized, as shown in Scheme 1, from the corresponding 2-benzopyrilium salt H by reaction with hydrazine hydrate, wherein X " is a counterion such as, for example perchlorate: J-
• the product 2,3 -benzodiazepine separates as a solid and is removed by filtration, washed with water and dried.
• the crude product may be purified by taking it up in a polar aprotic solvent such as dimethylformamide (DMF) at an elevated temperature, preferably 100-130°C, and decolorizing the solution with activated carbon. The carbon is removed by filtration and the filtered solution is diluted with water. The purified product precipitates out of the solution and is collected by filtration.
• a polar aprotic solvent such as dimethylformamide (DMF)
• intermediate benzopyrilium salt, H may be prepared from one of several starting materials. According to one such method, illustrated in Scheme 2, intermediate H is prepared from the corresponding aryl ethanol derivative D via the isochroman intermediate F. Another variation for preparing 2,3-benzodiazepines is illustrated in
• a substituted benzoic acid ester, A is dissolved in a suitable solvent, preferably ether and cooled to 0°C.
• a suitable solvent preferably ether and cooled to 0°C.
• Two equivalents of a suitable Grignard reagent are added dropwise and the reaction is allowed to warm to room temperature and monitored for disappearance of starting material.
• a proton source such as acetic acid.
• Volatiles are removed in vacuo, and the product B is used for the next step without purification.
• the ⁇ , ⁇ -substituted benzyl alcohol B is taken up in a high boiling solvent such as toluene and a catalytic amount of para-toluene sulfonic acid (p- TsOH).
• the mixture is warmed to reflux and may be monitored for disappearance of starting materials.
• the volatiles are removed in vacuo and the crude product C is purified by column chromatography.
• the substituted styrene C is hydroxylated under anti-Markovnikov conditions to give intermediate phenylethyl alcohol D.
• a solution of D, and of a suitably substituted benzaldehyde E (1.2 eq) are added to anhydrous dioxane.
• the resulting solution is then saturated with gaseous HCl and warmed, preferably to reflux temperature for about one hour.
• chromium trioxide (2g) in 35% sulfuric acid (20mL).
• the latter solution is added at a rate such that the reaction temperature remains below 5° C.
• the reaction mixture is allowed to rise to room temperature and is stirred at room temperature for two hours.
• the reaction mixture is then poured into water and extracted with an organic solvent, preferably ethyl acetate.
• the organic extract is washed with water and then with ice-cold 10% aqueous sodium hydroxide.
• the aqueous alkaline fraction is then acidified, preferably with dilute aqueous hydrochloric acid, and extracted with an organic solvent, preferably, chloroform.
• the chloroform extract is dried, filtered and concentrated under vacuum to give G.
• the crude residue may further be purified by column chromatography.
• the 2- ⁇ -acyl hydrocarbylbenzophenone G (5g) is dissolved in glacial acetic acid (15 mL). To this mixture is added 60% perchloric acid (7.5 mL). The resulting mixture is warmed to 100°C (steam bath) for three minutes. The mixture is allowed to cool to room temperature. Crystallization of the crude product may begin spontaneously at this point or may be induced by addition of ether or ethyl acetate.
• the product 2-benzopyrylium salt H is removed by filtration and purified by recrystallization, preferably from ethanol or glacial acetic acid/ethyl acetate.
• a 2,3-benzodiazepine derivative of formula I having an amine substituent the starting aromatic amine components must be protected with a protecting group or otherwise rendered unreactive in order for the amine to be rendered stable to the reaction conditions employed in the reaction schemes shown or referenced above.
• a means of circumventing the need for a protecting group may be to use a starting material containing an aromatic nitro group(s) in place of the desired aromatic amino group(s). The nitro group performs the same function as an amine protecting group in this synthesis and it may, following the synthesis steps that are incompatible with an amine substituent, be then reduced to an amine.
• Reduction of the aromatic nitro group can be done, for example, via catalytic hydrogenation.
• Catalytic hydrogenation provides the capability to selectively reduce the aromatic nitro group without reducing the olefin or other functionality in the intermediate.
• This synthetic strategy is disclosed in US Patent 4,614,740, wherein racemic 2,3- benzodiazepines were prepared with amino groups at a position corresponding the R 3 of formula I of the present invention. The entire disclosure of US 4,614,740 is incorporated herein by reference. Resolution of (i?)-2,3-Benzodiazepines of Formula I. The synthetic procedures shown (or referenced) above produce racemic mixtures of 2,3-benzodiazepines of formula I that are useful in methods of the /-
• Racemic 2,3-benzodiazepines of formula I may, for example, be converted to the (S)-dibenzoyltartaric acid salt, which is a diastereomeric mixture of SS and RS configurations.
• the pair of diastereomers (R,S) and (S,S) possess different properties, e.g., differential solubilities, that allow for the use of conventional separation methods. Fractional crystallization of diastereomeric salts from a suitable solvent is one such separation method. This resolution has been successfully applied to the resolution of racemic tofisopam. See
• racemic-2,3-benzodiazepines of formula I may be derivatized via, for example, acylation of an aryl hydroxy moiety, with a chiral acylating reagent, e.g., (S)-mandelic acid.
• a chiral acylating reagent e.g., (S)-mandelic acid.
• the resulting ester has a second chiral center, and thus exists as a diastereomeric pair separable using conventional methods such as crystallization or chromatography.
• the chiral moiety with which the racemic 2,3-benzodiazepine is derivatized may be removed.
• Racemic 2,3-benzodiazepines of formula I may be separated without diastereomer formation by differential absorption on a chiral stationary phase of a chromatography column, particularly a preparative HPLC column.
• Chiral HPLC columns are commercially available with a variety of packing materials to suit a broad range of separation applications.
• Exemplary stationary phases suitable for resolving the racemic 2,3-benzodiazepines of formula I include: (i) macrocyclic glycopeptides, such as silica-bonded vancomycin which contains 18 chiral centers surrounding three pockets or cavities; (ii) chiral ⁇ acid glycoprotein; (iii) human serum albumin; and (iv) cellobiohydrolase (CBH).
• Chiral ⁇ acid glycoprotein is a highly stable protein immobilized onto spherical silica particles that tolerates high concentrations of organic solvents, high and low pH, and high temperatures.
• Human serum albumin though especially suited for the resolution of weak and strong acids, zwitterionic and nonprotolytic compounds, has been used to resolve basic compounds.
• CBH is a very stable enzyme which has been immobilized onto spherical silica particles and is preferentially used for the separation of enantiomers of basic drugs from many compound classes.
• the present invention includes methods as described herein that use any and all observable conformations of compounds of formula I, preferably compounds having the (i?)-absolute configuration at carbon 5 of the benzodiazepine ring, which are biologically active in treatment or prevention of inflammatory disorders, particularly inflammatory disorders mediated by LTB 4 .
• compounds of formula I useful in the methods of the present invention may contain one or more chiral centers in addition to chiral center at the 5-position of the benzodiazepine ring of compounds of formula I.
• Such compounds may exist in, and may be isolated as pure enantiomeric or diastereomeric forms or as racemic mixtures.
• the present invention therefore includes methods that use any possible enantiomers, diastereomers, racemates or mixtures thereof of formula I (dictated by a chiral center other than the 5-position of the benzodiazepine ring) which are biologically active in the treatment or prevention of inflammatory disease states, particularly inflammatory disease states mediated by LTB 4 .
• Salts of Compounds of Formula I may take the form of pharmaceutically-acceptable salts.
• salts embraces salts commonly used to form alkali metal salts and to form addition salts of free acids or free bases.
• pharmaceutically-acceptable salt refers to salts that possess toxicity profiles within a range so as to have utility in pharmaceutical applications.
• Pharmaceutically unacceptable salts may nonetheless possess properties such as high crystallinity, which have utility in the practice of the present invention, such as for example utility in a synthetic process or in the process of resolving enantiomers from a racemic mixture.
• Suitable pharmaceutically-acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid. Examples of such inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phosphoric acid.
• organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, example of which are formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, mesylic, salicyclic, salicyclic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, 2-hydroxyethanesulfonic, toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, algenic, beta- hydroxybutyric, sali
• Suitable pharmaceutically acceptable base addition salts of compounds of formula I useful in methods of the invention include for example, metallic salts made from calcium, magnesium, potassium, sodium and zinc or organic salts made from NN-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. All of these salts may be prepared by conventional means from the corresponding compound of formula I by reacting, for example, the appropriate acid or base with the compound of formula I.
• Administration of Compounds of Formula I The compounds useful in methods of the invention may be administered to individuals (mammals, including animals and humans) afflicted with inflammatory disorders, particularly individuals afflicted with LTB 4 -mediated inflammatory disorders.
• the compounds useful in the practice of methods of the invention should be administered far enough in advance of a known event that increases the chance of an inflammatory disorder, particularly an inflammatory disorder mediated by LTB , such that the compound is able to reach the site of action in sufficient concentration to exert an effect.
• the pharmacokinetics of specific compounds may be determined by means known in the art and tissue levels of a compound in a particular individual may be determined by conventional analyses.
• One or more compounds useful in the practice of the present inventions may be administered simultaneously, or different compounds useful in the practice of the present invention may be administered at different times during treatment or prevention therapy.
• compounds of formula I may be administered for treatment of inflammatory disorders, in combination with one or more additional therapeutic agents.
• Such additional -agents include aminosalicylates, corticosteroids, antimetabolites, immunosuppressants, TNF- ⁇ inhibitors, inhibitors of leukotriene synthesis, and leukotriene antagonists; wherein when the additional therapeutic agents are leukotriene antagonist, they are other than compounds of formula I.
• Aminosalicylates believed useful in combination with compounds of formula I in methods of the invention include, for example, sulfasalazine and mesalamine.
• Corticosteroids believed useful in combination with compounds of formula I in methods of the invention include, for example, prednisone and budesonide.
• Antimetabolites believed useful in combination with compounds of formula I in methods of the invention include, for example, azathioprine.
• Immunosuppressants believed useful in combination with compounds of formula I in methods of the invention include, for example, cyclosporine and tacrolimus.
• TNF- ⁇ inhibitors believed useful in combination with compounds of formula I in methods of the invention include, for example, infliximab, etanercept, and adalimumab.
• Inhibitors of leukotriene synthesis believed useful in combination with compounds of formula I in methods of the invention include, for example, 5-LO inhibitors, e.g., ETH615, linetastine, lonapalene (RS43179), MK 886, and zileuton.
• Other inhibitors of leukotriene synthesis believed useful in combination with compounds of formula I include, for example, 15-HETE and leflunomide.
• Leukotriene antagonists useful in combination with compounds of formula I in methods of the invention include, for example, SC41930, SC53228, CGS-25019C, ONO-4057, SB-202247, VML295 (LY293111), CP-105696, CP- 195543, and U-75302. Routes of Administration.
• the compounds useful in methods of the invention may be administered for therapeutic effect by any route, for example enteral (e.g., oral, rectal, intranasal, etc.) and parenteral administration.
• Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intravaginal, intravesical (e.g., into the bladder), intradermal, topical or subcutaneous administration.
• the instillation of drug in the body of the patient in a controlled formulation with systemic or local release of the drug to occur at a later time.
• the drug may be localized in a depot for controlled release to the circulation, or controlled release to a local site of inflammation.
• the invention can be used, for instance, to prevent or treat an inflammatory skin disorder such as psoriasis, atopic dermatitis, and contact hypersensitivity.
• Other indications of interest include dry skin (sometimes called "winter itch").
• topical application of at least one of the compounds of Formula I e.g., one, two or three
• topical application of at least one of the compounds of Formula I will be often be indicated for some indications.
• Topical formulations in accord with the invention typically include a topical vehicle suitable for administration to the subject (particularly a mammal such as a human patient) in an amount suitable to reduce, inhibit or eliminate existing or potential skin irritation or inflammation. More specific formulations for topical use preferably less than about lOOmg/ml of one or more of the compounds provided above as Formula I, more preferably between from about O.OOlmg/ml to about 50 mg/ml, even more preferably between from about 0.01 mg/ml to about 10 mg/ml with about 0.1 mg/ml to about 5 mg/ml being suitable for many applications.
• the administered dose may be less than about 100 mg/day, more preferably less than about 50 mg/day, even more preferably less than about 10 mg or 5 mg/day and yet more preferably less than about 1 mg/day.
• Optimal topical concentrations of one or more of the compounds of Formula I can sometimes be adjusted (generally decreased) if one or more other anti-inflammatory component is included in the formulation.
• lower (eg., one-half less) amounts of compound may be used, while still maintaining comparable levels of anti-inflammation activity on the skin, by further including an approximately equal concentration of, for example a steroid or non-steroidal anti-inflammatory agent.
• a variety of suitable salts can be employed such as those mentioned above.
• Appropriate formulations generally suited for topical use include such vehicles (or vehicle components) as water; organic solvents such as alcohols (particularly lower alcohols readily capable of evaporating from the skin such as ethanol), glycols (such as glycerin), aliphatic alcohols (such as lanolin); mixtures of water and organic solvents (such as water and alcohol), and mixtures of organic solvents such as alcohol and glycerin (optionally also with water); lipid-based materials such as fatty acids, acylglycerols (including oils, such as mineral oil, and fats of natural or synthetic origin), phosphoglycerides, sphingolipids and waxes; protein-based materials such as collagen and gelatin; silicone-based materials (both non- volatile and volatile) such as cyclomethicone, demethiconol and dimethicon
• the vehicle may further include components adapted to improve the stability or effectiveness of the applied formulation, such as preservatives, antioxidants, skin penetration enhancers, sustained release materials, and the like.
• components adapted to improve the stability or effectiveness of the applied formulation such as preservatives, antioxidants, skin penetration enhancers, sustained release materials, and the like.
• preservatives such as preservatives, antioxidants, skin penetration enhancers, sustained release materials, and the like.
• vehicle components are well known in the art and are described in such reference works as Martindale ⁇ The Extra Pharmacopoeia (Pharmaceutical Press, London 1993) and Martin (ed.), Remington's Pharmaceutical Sciences.
• the choice of a suitable vehicle will depend on the particular physical form and mode of delivery that the formulation is to achieve.
• suitable forms include liquids (including dissolved forms of the cations of the invention as well as suspensions, emulsions and the like); solids and semisolids such as gels, foams, pastes, creams, ointments, "sticks” (as in lipsticks or underarm deodorant sticks), powders and the like; formulations containing liposomes or other delivery vesicles; rectal or vaginal suppositories, creams, foams, gels or ointments; and other forms.
• Typical modes of delivery for use as an anti-inflammatory for the skin include application using the fingers; application using a physical applicator such as a cloth, tissue, swab, stick or brush (as achieved for example by soaking the applicator with the formulation just prior to application, or by applying or adhering a prepared applicator already containing the formulation—such as a treated or premoistened bandage or patch, wipe, washcloth or stick—to the skin); spraying (including mist, aerosol or foam spraying); dropper application (as for example with ear or eye drops); sprinkling (as with a suitable powder form of the formulation); and soaking.
• a physical applicator such as a cloth, tissue, swab, stick or brush
• spraying including mist, aerosol or foam spraying
• dropper application as for example with ear or eye drops
• sprinkling as with a suitable powder form of the formulation
• oothing ingredients for optional use with the topical applications of the invention include, but are not limited to glycerin, aloe vera, chamomile, cola nitida extract, green tea extract, tea tree oil, licorice extract, allantoin, urea, caffeine or other xanthines, and glycyrrhizic acid and its derivatives may also be beneficially used with the invention to help reduce or block unwanted inflammation of the skin.
• a variety of particular administration routes can be employed such as those known to target the alimentary canal, stomach, small intestine or colon.
• Of particular interest is the delivery of the compounds intracolonically, for example by suppository or enema. See, for instance, Groning R, et al., Drug Dev. Ind Pharm, ID:527-39 (1984); Sheth, P. R, Drug Dev. Ind. Pharm. 10:313-39 (1983); Chien, Y. W., Drug Dev. Ind. Pharm 9:1291-330 (1983); Desai, S. and Bolton, S., Pharm. Res. 10: 1321-5 (1993)); Banakar (Amer. Pharm.
• 6,531,152 can be used to localize release of a desired agent in the gastrointestinal tract of the animal.
• a device includes a core that includes at least one of the compounds of Formula I.
• the compounds can be used as the sole active agent or as aheady discussed can be combined with one or more other agents to address the gastrointestinal indication.
• nearly any suitable amount of the compound can be used.
• the core diameter can range from 1 mm to 15 mm with a coating level ranging from 2 to 50 mg/cm 2 , for instance.
• formulations for targeting a compound of Formula I to the gastrointestinal tract typically contain preferably less than about lOOmg/ml of one or more of the compounds provided above as Formula I, more preferably between from about O.OOlmg/ml to about 50 mg/ml, even more preferably between from about 0.01 mg/ml to about 10 mg/ml with about 0.1 mg/ml to about 5 mg/ml being suitable for many applications.
• the administered dose may be less than 100 mg/day, more preferably less 50 mg/day, even more preferably less than 10 mg or 5 mg/day, and yet more preferably less than 1 mg/day of a compound of Formula I.
• the methods of the present invention may comprise administering 2,3- benzodiazepines, preferably (R)-2,3 -benzodiazepines, in the form of a pharmaceutical composition, in combination with a pharmaceutically acceptable carrier.
• the active ingredient in such formulations may comprise from 0.1 to 99.99 weight percent.
• pharmaceutically acceptable carrier is meant any carrier, diluent or excipient which is compatible with the other ingredients of the formulation and to deleterious to the recipient.
• the active agent is preferably administered with a pharmaceutically acceptable carrier selected on the basis of the selected route of administration and standard pharmaceutical practice.
• the active agent may be formulated into dosage forms according to standard practices in the field of pharmaceutical preparations.
• Suitable dosage forms may comprise, for example, tablets, capsules, solutions, parenteral solutions, troches, suppositories, or suspensions.
• the active agent may be mixed with a suitable carrier or diluent such as water, an oil (particularly a vegetable oil), ethanol, saline solution, aqueous dextrose (glucose) and related sugar solutions, glycerol, or a glycol such as propylene glycol or polyethylene glycol.
• Solutions for parenteral administration preferably contain a water-soluble salt of the active agent.
• Stabilizing agents, antioxidizing agents and preservatives may also be added.
• Suitable antioxidizing agents include sulfite, ascorbic acid, citric acid and its salts, and sodium EDTA.
• Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorbutanol.
• the composition for parenteral administration may take the form of an aqueous or nonaqueous solution, dispersion, suspension or emulsion.
• the active agent may be combined with one or more solid inactive ingredients for the preparation of tablets, capsules, pills, powders, granules or other suitable oral dosage forms.
• the active agent may be combined with at least one excipient such as fillers, binders, humectants, disintegrating agents, solution retarders, absorption accelerators, wetting agents absorbents or lubricating agents.
• the active agent may be combined with carboxymethylcellulose calcium, magnesium stearate, mannitol and starch, and then formed into tablets by conventional tableting methods.
• the compositions of the present invention can also be formulated so as to provide slow or controlled-release of the active ingredient therein, hi general, a controlled-release preparation is a composition capable of releasing the active ingredient at the required rate to maintain constant pharmacological activity for a desirable period of time.
• Such dosage forms can provide a supply of a drug to the body during a predetermined period of time and thus maintain drug levels in the therapeutic range for longer periods of time than other non-controlled formulations.
• U.S. Patent No. 5,674,533 discloses controlled-release compositions in liquid dosage forms for the administration of moguisteine, a potent peripheral antitussive.
• U.S. Patent No. 5,059,595 describes the controlled-release of active agents by the use of a gastro-resistant tablet for the therapy of organic mental disturbances.
• U.S. Patent No. 5, 591,767 discloses a liquid reservoir transdermal patch for the controlled administration of ketorolac, a non-steroidal anti-inflammatory agent with potent analgesic properties.
• U.S. Patent No. 5,073,543 discloses controlled-release formulations containing a trophic factor entrapped by a ganglioside-liposome vehicle.
• U.S. Patent No. 5,639,476 discloses a stable solid controlled-release formulation having a coating derived from an aqueous dispersion of a hydrophobic acrylic polymer.
• U.S. Patent No. 6,531,152 discloses, for instance, an immediate release gastrointestinal drug delivery system in which release of drugs in the gastrointestinal tract can be controlled in a location- and time-dependent manner. The patents cited above are incorporated herein by reference in their entirety.
• Biodegradable microparticles can be used in the controlled-release formulations of this invention.
• U.S. Patent No. 5,354,566 discloses a controlled-release powder that contains the active ingredient.
• U.S. Patent No. 5,733,566 describes the use of polymeric microparticles that release antiparasitic compositions. These patents are incorporated herein by reference.
• the controlled-release of the active ingredient can be stimulated by various inducers, for example pH, temperature, enzymes, water, or other physiological conditions or compounds.
• the controlled-release component can swell and form porous openings large enough to release the active ingredient after administration to a patient.
• controlled-release component in the context of the present invention is defined herein as a compound or compounds, such as polymers, polymer matrices, gels, permeable membranes, liposomes and/or microspheres, that facilitate the controlled-release of the active ingredient (e.g., (i?)-tofisopam or a pharmaceutically-acceptable salt thereof) in the pharmaceutical composition.
• the controlled-release component is biodegradable, induced by exposure to the aqueous environment, -4y-
• sol-gels can be used, wherein the active ingredient is incorporated into a sol-gel matrix that is a solid at room temperature.
• This matrix is implanted into a patient, preferably a mammal, having a body temperature high enough to induce gel formation of the sol-gel matrix, thereby releasing the active ingredient into the patient.
• the practice of the invention is illustrated by the following non-limiting examples. Examples Example 1: Synthesis of 1 -(3, 4-dimethoxyphenyl)-4-methyl-5-ethyl-7-hydroxy- 8-methoxy-5H-2,3-benzodiazepine.
• Example 2 Resolution of l-(3,4-dimethoxyphenyl)-4-methyl-5-ethyl-7- hydroxy-8-methoxy-5H-2,3-benzodiazepine to yield (i?)-l-(3,4-dimethoxy- phenyl)-4-methyl-5-ethyl-7-hydroxy-8-methoxy-5H-2,3-benzodiazepine. (i?,S)-l-(3,4-Dimethoxyphenyl)-4-methyl-5-ethyl-7-hydroxy-8-methoxy-
• 5H-2,3-benzodiazepine (43mg, dissolved in acetonitrile) is injected onto a Chirobiotic V column (ASTEAC, Whippany, NJ) Elution of the racemate with methyl-tert-butyl ether/acetonitrile 90/10 (v/v), at 40mL/minute, is monitored at 31 Onm, 2mm path.
• the R(+) enantiomer is the first compound to elute, and is collected and dried.
• the R(-), S(+), S(-) enantiomers, and some residual R(+) enantiomer coelute and are collected in subsequent fractions.
• Example 3 Synthesis of racemic- 1 -(3 -hydroxy-4-methoxyphenyl)-4-methyl- 5-ethyl-7,8-dimethoxy-5H-2,3-benzodiazepine Racemic-l-(3-hydroxy-4-methoxyphenyl)-4-methyl-5-ethyl-7,8- dimethoxy-5H-2,3-benzodiazepine was synthesized according to the route of Scheme 3.
• the mixture was heated at reflux for 3hr, saturated again with hydrogen chloride gas and allowed to stir at room temperature overnight. It was then poured into water, basified with dilute sodium hydroxide and extracted with methylene chloride. The combined methylene chloride extracts were dried and concentrated.
• Example 4 Synthesis of l-(3,4-dimethoxyphenyl)-4-methyl-5-ethyl-7- methoxy-8-hydroxy-5H-2,3-benzodiazepine Racemic l-(3,4-dimethoxyphenyl)-4-methyl-5-ethyl-7-methoxy-8- hydroxy-5H-2,3-benzodiazepine was synthesized according to the route of Scheme 4.
• the calculated values include 0.1M of residual ethyl acetate.
• Example 5 Resolution of l-(3-hydroxy-4-methoxyphenyl)-4-methyl-5-ethyl- 7,8-dimethoxy-5H-2,3-benzodiazepine ⁇
• the enantiomers of racemic-l-(3-hydroxy-4-methoxyphenyl)-4-methyl- 5-ethyl-7,8-dimethoxy-5H-2,3-benzodiazepine are resolved by chiral chromatography as follows. Racemic- 1 -(3-hydroxy-4-methoxyphenyl)-4-methyl-5-ethyl-7,8- dimethoxy-5H-2,3-benzodiazepine is loaded onto a semipreparative (500mm x
• Example 6 LTB 4 Binding Assay.
• the LTB 4 receptor binding activity of racemic tofisopam and enantiomerically pure (/?)- and (S)-tofisopam was determined via the guinea pig spleen membrane assay of Cheng et al, J. Pharmacol. Exp. Ther., 236(1), 126- 132, 1986. Reactions were carried out in a phosphate buffer (pH 7.4) containing NaCl, MgCl 2 , EDTA, and bacitracin.
• reaction volume 150 ⁇ L containing l.Omg/mL of the Guinea pig spleen membrane preparation and lnM [ 3 H]LTB 4 , with or without a competitor, was incubated at 0-4°C for 2 hours. Competitors included 2,3-benzodiazepines, and LTB 4 as a control.
• Competitors included 2,3-benzodiazepines, and LTB 4 as a control.
• the reaction was terminated by rapid vacuum filtration onto glass fiber filters. The filter was washed with cold buffer, dried and placed in a scintillation vial. Radioactivity trapped onto the filters was determined and compared to control values in order to ascertain any interactions of the test compound with the LTB 4 binding site. Data gathered in the binding experiments for test compounds and standards are -oy-
• Table 2 Summary of [ 3 H]LTB 4 binding data for (R)-, (S)- and racemic-l-(3,4-dimethoxyphenyl)-4-methyl-5-ethyI- 7,8-dimethox -5H-2,3-benzodiazepine
• the binding data shows that binding of racemic l-(3,4-dimethoxy- phenyl)-4-methyl-5-ethyl-7,8-dimethoxy-5H-2,3-benzodiazepine to the LTB 4 receptor is primarily due to the (R)-enantiomer, which binds with a Kj greater than 150x that of the (S)-enantiomer.
• Example 7 Effect of Tofisopam in a Rabbit Model of LTB 4 -induced Dermal Inflammation.
• Test Animals and Test Compounds Ten female New Zealand White Rabbits were assigned to dose groups as summarized in Table 3 below. Table 3: Test groups for the Rabbit model of LTB 4 - induced dermal inflammation.
• Test compounds were prepared as follows. The vehicle was first prepared by dissolving lOOmg of hydroxypropylmethylcellulose 2910 (HPMC)(Sigma Chemical, St. Louis, MO) in 50mL of 0.9% saline to yield a concentration of 2% HPMC. The three test articles, (R)-, (S)- and racemic tofisopam, were formulated by adding lg of each test article to lOmL of vehicle. B. Dosing and Intradermal Challenge.
• HPMC hydroxypropylmethylcellulose 2910
• the rabbits were sedated with ketamine/xylazine (35/5 mg/kg, s.c.) and an area of approximately 8x14 cm on the back was closely and carefully clipped to expose the skin, but not inflame or otherwise damage the epithelium.
• a grid of ten squares each approximately 2.5 x 2.5 cm was drawn on each animal's back using an indelible marker.
• the animals were then dosed intraperitoneally with the appropriate test article corresponding to the group. Thirty minutes after dosing with test or control article, the animals were challenged intradermally with the LTB 4 or LTB 4 -aminopropamide (LTB 4 -AP, a synthetic LTB 4 agonist) in the appropriate site as shown in Table 4.
• LTB 4 or LTB 4 -AP LTB 4 -aminopropamide
• Each injection site was marked with an indelible marker in order to identify the exact location of the intradermal injection site.
• Sixty minutes after challenge the animals were treated again with tofisopam or control articles.
• the animals were sacrificed 4 hrs. after challenge, and the intradermal injection sites were excised, fixed in 10% neutral buffered formalin (NBF), and submitted for histopathology. H&E-stained sections were read by a board-certified veterinary pathologist using light microscopy.
• Table 4 Intradermal injection site grid for test animals.
• D. Histopathology The marked skin sections were fixed for at least 48 hours on small cardboard squares. At gross trimming, three levels were cut through marked skin region in order to assure that the injection sites is brought into the plane of section. The skins were gross trimmed, processed by dehydration, embedded in paraffin, sectioned at 3-5 ⁇ m, and stained with hematoxylin and eosin. The tissues were evaluated histopathologically via light microscopy by a board-certified veterinary pathologist. Initially, the identity of the slides was masked in order to perform the initial evaluation and rank the slides. The slides were then unmasked and there was a careful assessment of the lesions and careful comparison of the tofisopam-treated sites to the vehicle and positive control tissues.
• the lesions were graded based upon the degree of inflammation and the degree of edema.
• Example 8 Dextran Sulfate Sodium Induced Colitis: Mouse Model of IBD.
• DSS dextran sulfate sodium
• This colitis was characterized by histological events and an influx of neutrophils, macrophages and mediators of inflammation similar to those observed with human inflammatory bowel diseases.
• drugs known to be of useful for treating IBD such as corticosteroids and 5-ASA, have been shown to have activity in this model. The following study was conducted in accordance with protocols of Okayasu et al, Gastroenterology, 98:694-702, 1990.
• test animals Female, 6 week old Swiss Webster mice, 18-30g were divided into ten groups, selected to eliminate any statistical differences in mean group weight. Each animal was dosed daily (IP) with either a test substance or vehicle, starting on Day 0. Beginning on Day 1, acute colon inflammation was induced by the administration ad libitum in drinking water of dextran sulfate sodium (DSS) as a 5% solution in tap water (lOmL/mouse/day for 5-6 days), with no other fluid source for animals in the DSS arm of the study. Filtered tap water was available ad libitum except for animals receiving 5% DSS as the sole source of fluid. After four days, signs of acute disease occurred with the loss of weight, diarrhea and bloody stools.
• IP dosed daily
• DSS dextran sulfate sodium
• Test animals were weighed daily from Day 0 to Day 8, or until completion of the study. The total duration of the study with DSS arm of the study was varied depending on the time progress of colitis. The condition of the test animals and consistency of stools was noted. At the conclusion of the study, test animals were euthanized (CO 2 ), a midline incision was made and a stool sample was obtained. The sample was placed on a slide and tested for occult blood (Quic-CultTM, Laboratory Diagnostics Co., Morganville, NJ). Occult blood was determined by placing two drops of the reagent onto the sample and observing any color change.
• Occult blood presence was graded using a scoring protocol assigning a score of 0 for no color; 1 for a very light blue color (+/-) forming in > 30 seconds; 2 for a blue color developing in 30 seconds or more (+); 3 for a change in color occurring in less than 30 seconds (++); and 4 for gross blood observable on the slide.
• the colon was gently stretched and the length from the colon-cecal junction to the end of the distal rectum was measured to the nearest 0.1cm.
• DAI Disease Activity Index
• DAI Disease Activity Index
• Table 8 Disease Activity Index for test animals in DSS-induced colitis stud .
• Example 9 Dextran Sulfate Sodium Induced Colitis- Effects of Low-Dosage R-Tofisopam. This study was conducted using a mouse model of inflammatory bowel disease as described by Okayasu et al (Gastroenterology 98:694-702, 1990) and modified by Murthy et al. (Digest. Dis. Sci. 38:1722-1734, 1993). i brief, the method involves feeding 5%) dextran sulfate sodium (DSS) in drinking water for 5-6 days to mice. After four days, signs of acute disease occur with the loss of weight, diarrhea and bloody stools.
• DSS dextran sulfate sodium
• Histological changes include initial shortening of the crypts, then areas of separation of the crypts and the muscularis mucosae in the absence of destructive inflammatory infiltrate. After five days, pathological changes become confluent with the appearance of erosions and early hyperplastic epithehum. Inflammation Scores are high with neutrophils, lymphocytes, and plasma cells in the lamina intestinal but sparing the epithelium. Agents given during this period are tested for potential prophylactic activity. Those given after the disease is established are used to evaluate therapeutic activity.
• mice mice/cage mice
• the animal room was temperature controlled and maintained on a 12-hour light/dark cycle. Following an acclimation period of 13 days, 60 healthy female mice were selected for the study.
• the test animals were distributed (10 mice/group) into each one of the six test groups as shown below.
• test compounds were suspended in a 0.5% w/w solution of carboxymethylcellulose (CMC) in distilled water. Each group of animals received the appropriate dose of the test compound starting on Day 0, as described above, by intracolonic (IC) administration using a ball tipped dosing needle and syringe at a daily dose volume of 0.2 ml mouse/day. Groups 2 through 6 were given a 5% w/w solution of dexatran sulfate sodium (DSS) in distilled water substituted for their normal water supply from Day 1 through the day before necropsy. Group 1 animals received water bottles containing water without DSS. No other source of fluids was available. Bottles were refilled daily with the appropriate volume of fresh DSS solution or water.
• CMC carboxymethylcellulose
• mice All mice were weighed on Day 0 and then daily from Days 5 through 9 and observed for signs of gross toxicity and behavioral changes, consistency of stool and presence of gross blood during the study. On Day 9, all surviving animals were euthanized by CO 2 inhalation and necropsied. Following euthanasia, a stool sample was obtained from the colon of each animal and was tested for occult blood (Quik- Cult Laboratory Diagnostics Co., Morganville, NJ). The colons were then removed and the length from the colo-cecal junction to the end of the distal rectum was measured. The whole colon was collected from each animal and preserved in 10% formalin. For each group, the disease activety index (DAI) was determined by evaluating changes in weight, Hemoccult positivity or gross bleeding, and stool consistency using the following system. Criteria for Scoring Disease Activity Index (DAI) 11
• Example 10 Effects of R-Tofisopam on Arachidonic Acid -Induced Inflammation Model in Mouse Ear.
• R-tofisospam were tested in a model of acute inflammation of the mouse ear produced by administration of arachidonic acid Several drugs have been shown to have anti-edematous effects in this model.
• the following study was conducted in accordance with the protocols of Burchart et al., Prostaglandins Leukot. Essent. Fatty Acids 1997 Apr; 56(4):301-306; Prostaglandins Leukot. Essent. Fatty Acids 1999 Jan;60(l):5-ll.
• mice 8-9 weeks of age ranging in weight from 20-24g, were group housed and acclimated for six days in a controlled environment (temperature 18-26°C; relative humidity 30-70%; 12 hours artificial light and 12 hours dark) in compliance with the National Research Council "Guide for the Care and Use of Laboratory Animals.” Temperature and humidity were monitored daily. All animals had access to drinking water and Certified Rodent Diet (TEKLAD) ad libitum and were monitored at least once daily for any abnormalities or for the development of infectious disease. Test compounds were prepared as follows. A solution of arachidonic acid (AA; Cat. No. A-9673, Sigma Chemical, St.
• Arachidonic acid at 2 mg/ 10 ⁇ l B Arachidonic acid at 2 mg/ 10 ⁇ l B. Experimental Design and Method of Analysis. The vehicle or test article was administered topically to the outer surface of both 5 the right and left ear in a volume of 10 ⁇ l/ear. Following a 30 minute ( ⁇ 5 minutes) absorption period, a 10 ⁇ l volume of AA/EtOH prepared as described above was administered topically to the inner surface of the ear. Thirty minutes ( ⁇ 5 minutes) after the administration of AA/EtOH, the animals were euthanized by CO 2 asphyxiation without exsanguination.
• Ears were measured for thickness 10 with a Mirutoyo micrometer and edema was calculated by subtracting the thickness measurement (in mm) of the control ear (right) from that of the test ear (left). Mean edema was calculated and compared using an ANOVA followed by a Tukey HSD Multiple Comparison Test ( ⁇ 0.05 Systat, v.9.01. C. Results. 15 All animals appeared normal during the course of the study. Mean animal data from the edema studies are presented in Table 14 and individual data are presented in Table 15.

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