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Title:
NOVEL METHODS
Document Type and Number:
WIPO Patent Application WO/2017/066729
Kind Code:
A1
Abstract:
Provided are novel methods for the treatment or prophylaxis of pain in a feline in need thereof comprising administering to the feline an effective amount of 1-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form.

Inventors:
MCKINNEY, Anthony (Inc.43 Thorndike Street,Suite S1-, Cambridge Massachusetts, 02141, US)
MURRAY, Frazer (Inc.43 Thorndike Street,Suite S1-, Cambridge Massachusetts, 02141, US)
Application Number:
US2016/057251
Publication Date:
April 20, 2017
Filing Date:
October 16, 2016
Export Citation:
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Assignee:
EBI LIFE SCIENCES, INC. (43 Thorndike Street, Suite S1-3Cambridge, Massachusetts, 02141, US)
International Classes:
C07D401/12; A61P3/00; A61P25/04; A61P25/20; A61P25/28; A61P43/00
Foreign References:
US20050282859A12005-12-22
Attorney, Agent or Firm:
JONES, Marya K. (Hoxie & Associates LLC, 75 Main StreetSuite 20, Millburn New Jersey, 07041, US)
Download PDF:
Claims:
CLAIMS

WHAT IS CLAIMED IS:

1. A method for the treatment or prophylaxis of pain, depression, and/or anxiety in a feline in need thereof comprising administering to the feline an effective amount of l-(4- methylphenyl)-3-azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form.

2. The method of claim 1 comprising administering 0.5 to 10 mg/kg/day, e.g., 1 to 10 mg/kg/day, e.g., 1 to 8 mg/kg/day, e.g., 1 to 6 mg/kg/day, e.g., 1 to 4 mg/kg/day, e.g., 1 to 2 mg/kg/day, e.g., 0.5 to 5 mg/kg/day, e.g., 0.5 to 2 mg/kg/day, e.g., 0.5 to 1 mg/kg/day, e.g., 1 mg/kg/day, of l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form.

3. The method of claim 1 or 2 comprising administering l-(4-methylphenyl)-3- azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form, once or twice daily.

4. The method of any one of claims 1-3, wherein l-(4-methylphenyl)-3- azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form, is crystalline.

5. The method of any one of claims 1-4, wherein l-(4-methylphenyl)-3- azabicyclo[3.1.0]hexane is in pharmaceutically acceptable salt form.

6. The method of any one of claims 1-5, wherein l-(4-methylphenyl)-3- azabicyclo[3.1.0]hexane in pharmaceutically acceptable salt form is an acid addition salt.

7. The method of any one of claims 1-6, wherein l-(4-methylphenyl)-3- azabicyclo[3.1.0]hexane in pharmaceutically acceptable salt form is l-(4-methylphenyl)-3- azabicyclo[3.1.OJhexane hydrochloride.

8. The method of claim 7, wherein l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane hydrochloride is l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane hydrochloride polymorph form B.

9. The method of claim 8, wherein l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane hydrochloride polymorph form B is substantially free of other polymorphic forms.

10. The method of claim 7, wherein l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane hydrochloride is l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane hydrochloride polymorph form A.

11. The method of claim 10, wherein l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane hydrochloride polymorph form A is substantially free of other polymorphic forms.

12. The method of any one of claims 1-7 comprising administering an effective amount of (+)-l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form.

13. The method of any one of claims 1-7 comprising administering an effective amount of (-)-l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form.

14. The method of any one of claims 1-13 further comprising administering one or more of a local anesthetic, an opioid, an alpha-2 agonist, a non-steroidal anti-inflammatory drug (NSAID), a corticosteroid, a N-methyl-D-aspartate (NMD A) receptor antagonist, a NKl receptor antagonist, a calcium channel blocker, a muscle relaxant, and a bisphosphonate.

15. The method of claim 14, wherein the administration with l-(4-methylphenyl)-3- azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form, is concurrent.

16. The method of claim 14, wherein the administration with l-(4-methylphenyl)-3- azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form, is sequential.

17. The method of any one of claims 1-16, wherein the pain is acute pain.

18. The method of any one of claims 1-16, wherein the pain is chronic pain.

19. The method of any one of claims 1-18, wherein the pain is neuropathic pain.

20. The method of any one of claims 1-19, wherein the feline is a domestic cat.

Description:
NOVEL METHODS

[0001] This application claims the benefit of U.S. Provisional Application No. 62/242,940 filed October 16, 2015, which is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

[0002] The present disclosure relates to novel methods for the treatment or prophylaxis of pain, depression, and/or anxiety in a feline in need thereof comprising administering to the feline an effective amount of l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form.

BACKGROUND

[0003] Pain and anxiety management are important medical problems facing cats as advances in veterinary medicine and nutrition prolong their lifetimes. More recently, cats are living far into old age, and, as such, non-life-threatening diseases or disorders that may cause substantial pain and depression in their lives are increasingly plaguing them. Chronic pain is an especially important concern. However, developing effective pain medication for cats has proven challenging. Analgesics must be used with caution in cats.

[0004] For instance, cats have a reduced ability to break down NSAIDs and are more sensitive to the drugs' side effects. Commonly used NSAIDs such as aspirin, ibuprofen, naproxen, and other aspirin substitutes may be toxic in cats. Currently, there are no non-steroidal anti-inflammatory drugs (NSAIDs) approved for long-term use in cats due to safety concerns. Meloxicam is approved for cats as a one-time-only injection to control pain and inflammation after spaying, neutering, and orthopedic surgery. Repeated use of meloxicam in cats is associated with sudden- onset kidney failure and death. Robenacoxib is also approved for cats to control pain and inflammation after spaying, neutering, and orthopedic surgery; the tablets are given by mouth for no more than three days.

[0005] In addition, cats should not be given acetaminophen because they lack certain enzymes that the liver needs to safely break down the drug. And, phenylbutazone, an analgesic prescribed for horses, dogs, and other animals may produce toxicity like aspirin and acetaminophen when used in cats. [0006] Opioids may also be used to manage pain in felines, however, opioids have a number of disadvantages as well. Problems with opioids include the special licensing and record keeping required and the risk of diversion. In addition, the effect of meperidine, morphine, and codeine in cats is unpredictable. For instance, morphine, in a dose appropriate for a small dog, produces apprehension, excitability, and drooling in the cat. When this minimum dose is exceeded, the cat may convulse and die.

[0007] The metabolic differences between felines and other species, for example humans and canines, has made development of novel therapeutic agents in humans and other animals difficult to translate to the feline setting.

[0008] Thus, there is a need for novel methods that may be used to treat pain in felines.

BRIEF SUMMARY

[0009] Provided is a method of treating pain in a feline in need thereof comprising administering to the feline an effective amount of l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane (bicifadine), in free or pharmaceutically acceptable salt form.

[0010] Also provided is a method of treating depression in a feline in need thereof comprising administering to the feline an effective amount of l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane (bicifadine), in free or pharmaceutically acceptable salt form.

[0011] Also provided is a method of treating anxiety (e.g., separation anxiety and/or generalized anxiety disorder) in a feline in need thereof comprising administering to the feline an effective amount of l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane (bicifadine), in free or

pharmaceutically acceptable salt form.

[0012] Further areas of applicability of the present disclosure will become apparent from the detailed description provided hereinafter. It should be understood that the detailed description and specific examples, while indicating preferred embodiments, are intended for purposes of illustration only and are not intended to limit the scope of the disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 depicts time course of plasma levels of bicifadine (1, 5, and 10 mg/kg po) after administration of bicifadine to female (F) and male (M) cats. [0014] Figure 2 depicts time course of plasma levels of bicifadine (1 and 5 mg/kg po) after administration of bicifadine to female (F) and male (M) cats.

[0015] Figure 3 depicts time course of plasma levels of bicifadine (5 mg/kg po) and lactam metabolite after administration of bicifadine to female (F) and male (M) cats.

DETAILED DESCRIPTION

[0016] As used throughout, ranges are used as shorthand for describing each and every value that is within the range. Any value within the range can be selected as the terminus of the range.

[0017] All references cited herein are hereby incorporated by reference in their entireties. In the event of a conflict in a definition in the present disclosure and that of a cited reference, the present disclosure controls.

[0018] l-(4-methylphenyl)-3-azabicyclo[3.1. OJhexane, bicifadine, of Formula I below, is described in U.S. Patent Nos. 4,131,611 and 4,435,419 and U.S. Patent Publication No.

2013/0345280, all of which are hereby incorporated by reference in their entireties.

Formula I

[0019] U.S. Patent No. 7,094,799 describes l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane hydrochloride as a non-narcotic analgesic. However, the use of l-(4-methylphenyl)-3- azabicyclo[3.1.OJhexane to treat pain in a feline in need thereof has not been previously disclosed.

[0020] l-(4-methylphenyl)-3-azabicyclo[3.1. OJhexane is safe and well-tolerated in humans.

However, dogs receiving 10-60 mg/kg doses of l-(4-methylphenyl)-3-azabicyclo[3.1. OJhexane show signs of toxicity (see Example 4). Unexpectedly, while other pain medications that are safe and effective in humans and dogs have not proved safe for cats, it has been discovered that l-(4- methylphenyl)-3-azabicyclo[3.1. OJhexane is tolerated in cats at levels of up to 10 mg/kg. l-(4- methylphenyl)-3-azabicyclo[3.1. OJhexane has a surprisingly good side effect profile in cats compared to l-(4-methylphenyl)-3-azabicyclo[3.1. OJhexane in other species. [0021] l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane is a serotonin-norepinephrine-dopamine reuptake inhibitor. In felines, l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane is believed to be metabolized with a dual extrahepatic (MAO-B) and hepatic (cytochrome) metabolism. This dual non-renal mechanism of metabolism may relieve stress on the kidneys. Further, because l-(4- methylphenyl)-3-azabicyclo[3.1.0]hexane may not significantly inhibit cyclooxygenase in felines, use of l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane may be less detrimental to the stomach and colon compared to NSAIDs. Moreover, l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane's E (norepinephrine) and 5-HT (serotonin) reuptake activity may benefit felines that are anxious or depressed because of injury, pain, or other disease.

[0022] Provided herein is a method of treating pain in a feline in need thereof comprising administering to the feline an effective amount of l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form.

[0023] l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane may be synthesized as described in U.S. Patent Nos. 4, 131,611 and 4,435,419.

[0024] As used herein, the singular forms "a," "an," and "the" include the plural reference unless the context clearly indicates otherwise.

[0025] As used herein, "l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane" is to be understood as embracing the compound in any form, for example, free or pharmaceutically acceptable salt form, e.g., as a pharmaceutically acceptable acid addition salt. Pharmaceutically acceptable salts are known in the art and include salts that are physiologically acceptable at the dosage amount and form to be administered, for example, hydrochloride salts.

[0026] "l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane" is also to be understood as embracing its enantiomers, crystalline and amorphous forms, and mixtures thereof. "Crystalline form" and "polymorph" may be used interchangeably herein, and are meant to include all crystalline forms of l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form, including, for example, solvates (including hydrates).

[0027] The (+) and (-) enantiomers of l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane are described by Epstein et al., Journal of Medicinal Chemistry, 1981, 24 (5), 481. See also U.S. Patent Nos. 4, 131,611, 4,231,935, and 4,435,419. In some embodiments, an effective amount of (+)-l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form, is used. In some embodiments, an effective amount of (-)-l-(4-methylphenyl)-3- azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form, is used. In some embodiments, a mixture of (+)-l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form, and (-)-l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form, is used (e.g., a racemic mixture, a mixture comprising > 50% (+)-l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane, in free or

pharmaceutically acceptable salt form, or a mixture comprising > 50% (-)-l-(4-methylphenyl)-3- azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form).

[0028] In some embodiments, an effective amount comprises essentially pure (+)-l-(4- methylphenyl)-3-azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form (e.g., having 90-95% of (+)-l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane, in free or

pharmaceutically acceptable salt form, by weight of total l-(4-methylphenyl)-3- azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form, present), essentially pure (-)-l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form, or any mixture of (+)-l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form, and (-)-l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form.

[0029] l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane hydrochloride exists in at least two polymorphic forms, labeled polymorphs A and B as disclosed in U.S. Patent No. 7,094,799, which is hereby incorporated by reference in its entirety. Crystalline and amorphous forms of 1- (4-methylphenyl)-3-azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form, may be used in any combination or in forms that are substantially free of one or more of the other crystalline forms or free of the amorphous form.

[0030] Thus, useful forms of l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane within the methods, uses, and compositions described herein include l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form, as well as enantiomers, polymorphs, solvates, hydrates, prodrugs, and combinations thereof.

[0031] "l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane" also encompasses its stable and unstable isotopes. Stable isotopes are nonradioactive isotopes that contain one additional neutron compared to the abundant nuclides of the same species (i.e., element). For example, the hydrogen atom at a certain position on l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane may be replaced with deuterium (a stable isotope which is non-radioactive). Alternatively, unstable isotopes, which are radioactive isotopes that contain additional neutrons compared to the abundant nuclides of the same species (i.e., element), e.g., 14 C, may replace the corresponding abundant species.

[0032] In some embodiments, an effective amount comprises at least 10% to 10-20%>, 20-35%, 35-50%, 50-70%, 70-85%, 85-95%, and up to 95-99% or greater (by weight) polymorph B.

[0033] As used herein, "substantially free of other polymorphic forms" means that the crystalline material, e.g., polymorph A or polymorph B, contains 10% w/w or less of any other crystalline form. For example a crystalline material, e.g., polymorph A or polymorph B, contains 5% w/w or less of any other crystalline form, e.g., 1% w/w or less of any other crystalline form.

[0034] As used herein, "treatment" and "treating" include minimizing or eliminating pain in a feline.

[0035] As used herein, "effective amount" is intended to encompass a therapeutically effective amount to treat a specific disease or disorder.

[0036] As used herein, "feline" refers to animals belonging to the family Felidae including, for example, the domestic cat (also referred to as cat herein), tigers, lions, cougars, cheetahs, lynxes, ocelots, jaguars, leopards, and snow leopards.

[0037] As used herein, "pain" includes pain of varying severity, i.e. mild, moderate, and severe pain, as well as acute and chronic pain.

[0038] In some embodiments, pain occurs because of an injury (e.g., an orthopedic injury, e.g., a bone fracture or contusion), degenerative issues in the animal's tissues, blunt trauma, or following surgery or medical treatment (e.g., spaying, neutering, or declawing). In some embodiments, pain occurs because of a soft tissue injury, such as an injury to a ligament or tendon. In some embodiments, the pain is associated with colic in the feline. As used herein, "colic" encompasses all forms of gastrointestinal conditions which cause pain as well as other causes of abdominal pain not involving the gastrointestinal tract. Colic may include a visceral component. In some embodiments, the pain is due to arthritis, for example osteoarthritis (also known as degenerative joint disease).

[0039] As used herein, "acute pain" refers to either an initial phase of a painful condition that either largely resolves within several hours, days, or months (typically lasting no more than 3 months) or progresses on to a subacute pain (e.g., lasting 3-6 months) or chronic pain (e.g., persisting, in some cases intermittently, for more than 3 months, and often more than 6 months). Acute pain also refers to a transient exacerbation or flare up of a chronic pain condition in which pain intensity worsens substantially, whereby supplemental treatment and/or upwards dose adjustment is indicated, provided that such treatment would be tolerated adequately.

[0040] Acute pain may occur with or after colic, trauma, fracture, surgery (e.g., spaying, neutering, or declawing), infection, or inflammatory disease.

[0041] As used herein, "chronic pain" refers to pain that lasts more than 3 months, for example more than 6 months, and/or extends beyond the expected period of healing. Chronic pain may be considered pathologic, or having the characteristics of a diseased state. It may be unrelenting, have no identifiable cause, spread beyond the original site of an injury or insult, and serve no biological function.

[0042] Chronic pain may occur with musculoskeletal injury or disease (e.g., chronic pain may occur with osteoarthritis, stomatitis, and intervertebral disk disease). Chronic pain may also include pain consequent to cancer, diabetes, and chronic low back pain.

[0043] As used herein, "neuropathic pain" refers to pain caused by injury or damage to either the peripheral and/or the central nervous system.

[0044] Felines with injuries that involve nerve damage, felines that have had neurectomies, and felines with back pain may suffer from neuropathic pain. Neuropathic pain may follow onychectomy or limb or tail amputation.

[0045] As used herein, "concurrently" means the compounds are administered simultaneously or within the same composition. In some embodiments, the compounds are administered simultaneously. In some embodiments, the compounds are administered within the same composition.

[0046] Provided herein is a method (Method 1) for the treatment or prophylaxis of pain in a feline in need thereof comprising administering to the feline an effective amount of l-(4- methylphenyl)-3-azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form.

[0047] Also provided herein is a method (Method 2) for the treatment or prophylaxis of depression in a feline in need thereof comprising administering to the feline an effective amount of l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form.

[0048] Also provided herein is a method (Method 3) for the treatment or prophylaxis of anxiety (e.g., separation anxiety and/or generalized anxiety disorder) in a feline in need thereof comprising administering to the feline an effective amount of l-(4-methylphenyl)-3- azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form.

[0049] Further provided is any one of Methods 1, 2, or 3, e.g., Method 1, e.g., Method 2, e.g., Method 3, as follows:

1.1 Any one of Methods 1, 2, or 3, e.g., Method 1, e.g., Method 2, e.g., Method 3, comprising administering 0.5 to 10 mg/kg/day, e.g., 1 to 10 mg/kg/day, e.g., 1 to 8 mg/kg/day, e.g., 1 to 6 mg/kg/day, e.g., 1 to 4 mg/kg/day, e.g., 1 to 2 mg/kg/day, e.g., 0.5 to 5 mg/kg/day, e.g., 0.5 to 2 mg/kg/day, e.g., 0.5 to 1 mg/kg/day, e.g., 1 mg/kg/day, of l-(4- methylphenyl)-3-azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form. Preferably, a dose of 1 mg/kg animal weight per day is administered. Orally administered l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane has surprisingly high plasma levels in cats, with the C max and AUC for a cat following a 5 mg/kg oral dose being higher than, for example, the C max and AUC for a rat receiving a 20 mg/kg oral dose. For instance, Example 1 shows C max and AUC for cats after a 5 mg/kg oral dose is 7546 ng/ml and 25388 ng ' h/ml, respectively, while Musick et al. reported C max and AUC for female rats after a 20 mg/kg oral dose was 3530 ng/ml and 7440 ng ' h/ml, respectively, and Cmax and AUC for male rats after a 20 mg/kg oral dose was 2620 ng/ml and 5470 ng ' h/ml (Musick, T. et al., "Pharmacokinetics, Disposition, and Metabolism of Bicifadine in the Mouse, Rat, and Monkey," Drug Metabolism and Disposition, 2008, 36 (2), 241- 251, which is hereby incorporated by reference in its entirety). Basile et al. reported that the Cmax after oral administration of a single dose of 6 or 20 mg/kg to male rats was 483 and 1517 ng/ml, respectively (Basile, A. et al., "Characterization of the Antinociceptive Actions of Bicifadine in Models of Acute, Persistent, and Chronic Pain," The Journal of Pharmacology and Experimental Therapeutics, 2007, 321 (3), 1208-1225, which is hereby incorporated by reference in its entirety). Basile et al. also reported that oral dosages of bicifadine in that range were effective in rats in models of acute, persistent, and chronic pain. Thus, the bioavailability of l-(4-methylphenyl)-3- azabicyclo[3.1.0]hexane in felines may be higher (and the safe and effective dose correspondingly lower) compared to other species. Any one of Methods 1, 2, or 3, e.g., Method 1, e.g., Method 2, e.g., Method 3, or 1.1 comprising administering l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form, once or twice daily.

Any one of Methods 1, 2, or 3, e.g., Method 1, e.g., Method 2, e.g., Method 3, or 1.1 or 1.2 wherein l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form, is crystalline.

Any one of Methods 1, 2, or 3, e.g., Method 1, e.g., Method 2, e.g., Method 3, or 1.1-1.3 wherein l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane is in pharmaceutically acceptable salt form.

Any one of Methods 1, 2, or 3, e.g., Method 1, e.g., Method 2, e.g., Method 3, or 1.1-1.4 wherein l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane in pharmaceutically acceptable salt form is an acid addition salt.

Any one of Methods 1, 2, or 3, e.g., Method 1, e.g., Method 2, e.g., Method 3, or 1.1-1.5 wherein l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane in pharmaceutically acceptable salt form is l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane hydrochloride.

Method 1.6 wherein l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane hydrochloride is 1- (4-methylphenyl)-3-azabicyclo[3.1.0]hexane hydrochloride polymorph form B.

Method 1.7 wherein l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane hydrochloride polymorph form B is substantially free of other polymorphic forms.

Method 1.6 wherein l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane hydrochloride is 1- (4-methylphenyl)-3-azabicyclo[3.1.0]hexane hydrochloride polymorph form A.

Method 1.9 wherein l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane hydrochloride polymorph form A is substantially free of other polymorphic forms.

Any one of Methods 1, 2, or 3, e.g., Method 1, e.g., Method 2, e.g., Method 3, or 1.1-1.6 comprising administering an effective amount of (+)-l-(4-methylphenyl)-3- azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form.

Any one of Methods 1, 2, or 3, e.g., Method 1, e.g., Method 2, e.g., Method 3, or 1.1-1.6 comprising administering an effective amount of (-)-l-(4-methylphenyl)-3- azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form.

Any one of Methods 1, 2, or 3, e.g., Method 1, e.g., Method 2, e.g., Method 3, or 1.1-1.12 further comprising administering one or more of a local anesthetic, an opioid, an alpha-2 agonist, a non-steroidal anti-inflammatory drug (NSAID), a corticosteroid, a N-methyl- D-aspartate (NMD A) receptor antagonist, a NKl receptor antagonist, a calcium channel blocker, a muscle relaxant, and a bisphosphonate.

Any one of Methods 1, 2, or 3, e.g., Method 1, e.g., Method 2, e.g., Method 3, or 1.1-1.13 further comprising administering one or more of procaine, chloroprocaine, mepivacaine, prilocaine, lidocaine, articaine, tetracaine, bupivacaine (with or without epinephrine), levobupivacaine, ropivacaine, capsaicin, resiniferatoxin, morphine, oxymorphone, meperidine, methadone, hydromorphone, fentanyl, butorphanol, codeine, hydrocodone, tramadol, buprenorphine, alfentanil, sufentanil, pentazocine, nalbuphine, naloxone, nalmefene, xylazine, detomidine, medetomidine, dexmedetomidine, romifidine, aspirin, flunixin (e.g., flunixin meglumine), ketoprofen, firocoxib, diclofenac, meloxicam, piroxicam, carprofen, vedaprofen, robenacoxib, tolfenamic acid, ketorolac, prednisone, prednisolone, hydrocortisone, triamcinolone (e.g., triamcinolone acetonide and

triamcinolone diacetate), methylprednisolone, dexamethasone, fludrocortisone, isoflupredone, flumethasone, betamethasone, ketamine, amantadine, tiletamine, maropitant, gabapentin, methocarbamol, imipramine, amitriptyline, duloxetine, sarapin, atropine, hyoscine N-butylbromide, dipyrone, hyoscine, and pamidronate.

Any one of Methods 1, 2, or 3, e.g., Method 1, e.g., Method 2, e.g., Method 3, or 1.1-1.14 comprising administering one or more of another anti-depressant or anxiolytic.

Any one of Methods 1, 2, or 3, e.g., Method 1, e.g., Method 2, e.g., Method 3, or 1.1-1.15 further comprising performing one or more of acupuncture, rehabilitation, low-level laser, transcutaneous electric nerve stimulation, hydrotherapy, massage, prolotherapy, neural therapy, and reflexology.

Any one of Methods 1, 2, or 3, e.g., Method 1, e.g., Method 2, e.g., Method 3, or 1.1-1.16 further comprising administering one or more of a nutraceutical and an herbal supplement. Any one of Methods 1.13, 1.14, 1.15, or 1.17 wherein the administration with l-(4- methylphenyl)-3-azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form, is concurrent.

Any one of Methods 1.13, 1.14, 1.15, or 1.17 wherein the administration with l-(4- methylphenyl)-3-azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form, is sequential. Any one of Methods 1 or 1.1-1.19 wherein the pain is acute pain.

Any one of Methods 1 or 1.1-1.19 wherein the pain is chronic pain.

Any one of Methods 1 or 1.1-1.21 wherein the pain is neuropathic pain.

Any one of Methods 1 or 1.1-1.22 wherein the pain is co-morbid with depression.

Any one of Methods 1 or 1.1-1.23 wherein the pain is co-morbid with anxiety.

Any one of Methods 3 or 1.1-1.19 or 1.24 wherein the anxiety is separation anxiety. Any one of Methods 3 or 1.1-1.19 or 1.24 wherein the anxiety is generalized anxiety disorder.

Any one of Methods 3 or 1.1-1.19, 1.25, or 1.26 wherein the anxiety is co-morbid with pain.

Method 1.27 wherein the pain is acute pain.

Method 1.27 wherein the pain is chronic pain.

Any one of Methods 1.27-1.29 wherein the pain is neuropathic pain.

Any one of Methods 2 or 1.1-1.19 wherein the depression is co-morbid with pain.

Method 1.31 wherein the pain is acute pain.

Method 1.31 wherein the pain is chronic pain.

Any one of Methods 1.31-1.33 wherein the pain is neuropathic pain.

Any one of Methods 1, 2, or 3, e.g., Method 1, e.g., Method 2, e.g., Method 3, or 1.1-1.34 wherein the feline is a domestic cat.

Any one of Methods 1, 2, or 3, e.g., Method 1, e.g., Method 2, e.g., Method 3, or 1.1-1.35 wherein the feline is male.

Any one of Methods 1, 2, or 3, e.g., Method 1, e.g., Method 2, e.g., Method 3, or 1.1-1.35 wherein the feline is female.

Any one of Methods 1, 2, or 3, e.g., Method 1, e.g., Method 2, e.g., Method 3, or 1.1-

1.37, wherein the l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane, in free or

pharmaceutically acceptable salt form, is administered as a pharmaceutical composition comprising an effective amount of l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form, in combination with a pharmaceutically acceptable diluent or carrier.

Any one of Methods 1, 2, or 3, e.g., Method 1, e.g., Method 2, e.g., Method 3, or 1.1-

1.38, wherein the l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form, is administered as a pharmaceutical composition consisting essentially of an effective amount of l-(4-methylphenyl)-3- azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form, in combination with a pharmaceutically acceptable diluent or carrier.

[0050] Where one or more active agents and l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form, are administered, the active agent(s) and l-(4- methylphenyl)-3-azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form, may be administered concurrently or sequentially in any order.

[0051] Also provided herein is use of l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form, for the treatment or prophylaxis of pain, depression, and/or anxiety in a feline in need thereof, for example, for use in any one of Methods 1, 2, or 3, e.g., Method 1, e.g., Method 2, e.g., Method 3, e.g., any one of Methods 1.1-1.39.

[0052] Also provided herein is use of l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form, in the manufacture of a medicament for the treatment or prophylaxis of pain, depression, and/or anxiety in a feline in need thereof, for example, for use in any one of Methods 1, 2, or 3, e.g., Method 1, e.g., Method 2, e.g., Method 3, e.g., any one of Methods 1.1-1.39.

[0053] Also provided is a pharmaceutical composition comprising an effective amount of l-(4- methylphenyl)-3-azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form, in combination with a pharmaceutically acceptable diluent or carrier, for use in the treatment or prophylaxis of pain, depression, and/or anxiety in a feline in need thereof, for example, for use in any one of Methods 1, 2, or 3, e.g., Method 1, e.g., Method 2, e.g., Method 3, e.g., any one of Methods 1.1-1.39.

[0054] Also provided is a pharmaceutical composition consisting essentially of an effective amount of l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form, in combination with a pharmaceutically acceptable diluent or carrier, for use in the treatment or prophylaxis of pain, depression, and/or anxiety in a feline in need thereof, for example, for use in any one of Methods 1, 2, or 3, e.g., Method 1, e.g., Method 2, e.g., Method 3, e.g., any one of Methods 1.1-1.39.

[0055] A dose or method of administration of the dose of the present disclosure is not particularly limited. Dosages employed will vary depending, e.g., on the particular disease or condition to be treated, the mode of administration, and the therapy desired. l-(4-methylphenyl)- 3-azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form, may be

administered by any suitable route, including orally, parenterally, transdermally, or by inhalation, although various other known delivery routes, devices and methods can likewise be employed.

[0056] Pharmaceutical compositions comprising l-(4-methylphenyl)-3-azabicyclo[3.1.0]hexane, in free or pharmaceutically acceptable salt form, may be prepared using conventional diluents or excipients and techniques known in the galenic art. Thus, dosage forms may include tablets, capsules, solutions, suspensions, pastes, and the like.

EXAMPLES

Example 1

[0057] Eight cats are acclimated to study conditions for at least 6 days during which they are subject to physical examinations; body weight measurements; blood collection for hematology and clinical chemistry; daily clinical observations; sham dosing procedures; and mock blood collections.

[0058] Cats are Felis catus, domestic short haired cat. Physiologically the cats are healthy.

Female cats are non-pregnant and non-lactating. Cats are between 11 and 30 months of age as of the day of dosing. Body weight of cats is between 3.4 and 5.1 kg on Day -3 (these body weights are used for dose calculation).

[0059] Cats are not treated with drugs within the previous 28 days, with the exception of anesthetics used for vascular access port placement or analgesics used pre or post-surgery and sedatives prior to shaving for blood collection site preparation.

[0060] Cats are offered 1 cup (approximately 150 g) of feed each day. Water is available ad libitum.

[0061] Four treatment groups of 2 cats each (1 male/1 female) are used in the study. Each cat is orally dosed with an individually customized FIPMC (hydroxylpropylmethylcellulose) capsule (containing the total drug amount calculated based on body weight and dose group) as below in Table 1 : Table 1.

[0062] Capsules are prepared as follows using an analytical balance accurate to at least 0.01 mg:

• The dose is calculated for each cat based on Day -3 body weight;

• Correction from base to salt form of test article is made (1.0 gram of bicifadine

hydrochloride is equivalent to 0.8261 g of bicifadine), and the test article weight of salt form to be added in the capsule is calculated;

• The empty capsule (both parts, body and cap) is weighed;

• The target test article amount (±0.1 mg) is weighed directly into the capsule body;

• The weight difference between the filled and empty capsule is calculated. Acceptable weights are ±5% of test article calculated dose;

• Plastic vials are labeled with Study Animal ID, group ID, test article name, lot number, study number, and capsule number;

• Vials are stored in dark at USP controlled temperature (actual storage temperature

between 20.5 and 22.4°C).

[0063] No cats are removed from the study during the treatment phase.

[0064] The cats are monitored for behavioral effects by blinded observers.

[0065] Cats in Group 1 and 2 receive bicifadine at 1 and 10 mg/kg, respectively, by oral administration once daily for 4 consecutive days.

[0066] Group 3 cats are dosed on the first day at 30 mg/kg. Because moderate toxicity is observed after the first dose, the dose is reduced to 5 mg/kg once daily for the subsequent three days.

[0067] For the purpose of pharmacokinetic (PK) evaluation, blood samples are collected from all cats of Groups 1 to 3 (doses of 1, 10, and 30 mg/kg) prior to (collect one day prior to dosing) and following the first dose at 15 min (± 2 min), 30 min (± 2 min), 45 min (± 2 min), and 1 (± 2 min), 1.5 (± 2 min), 2 (± 5 min), 3 (± 5 min), 4 (± 5 min), 6 (± 5 min), 8 (± 5 min), 12 (± 5 min), and 24 (± 5 min) hours (h). Due to the changes in dose schedule a fourth group of 2 cats (one male and one female) is dosed only once on Day 2 at 5 mg/kg for PK evaluation as above.

[0068] On the first dosing day, cats are observed prior to dosing and at 30 min (± 5 min) and 1, 2, 4, 6, 8, and 12 h (± 10 min) post dosing.

[0069] For the remainder of the study, cats are observed prior to dosing and at 1, 2, and 4 h (± 10 min) post dose with the exception of Day 1, where clinical observations for the 4 h time point are performed 37 to 40 min later than the nominal time.

[0070] Clinical observations assess the eyes, respiration, mucous membranes, femoral pulses, somatomotor status, fecal consistency and presence of blood, and behavior.

[0071] The two cats in Group 4 are observed only on Day 2 prior to dosing and at 1, 2, and 4 and 24 h (± 10 min) post dose.

[0072] Body weight is recorded daily on Days 1 to 4.

[0073] Cats are fasted at least 5 h prior to blood collection for clinical chemistry and

hematology.

[0074] Blood for both hematology and clinical chemistry evaluation is collected from each cat for Groups 1 to 3 on Day 0 at 4 h (± 15 min) after dosing (clinical chemistry only), on Day 1 (24 h (± 1 h) after the first dose), and Day 4.

[0075] Cats receiving a dose of 30 mg/kg bicifadine show marked mydriasis with poor or lack of pupillary light reflex; changes in behavior and hyperaesthesia; moderate hypersalivation (female cat only); increased heart rate (>240 beats/min); and respiratory distress (associated with respiratory rate >240 breaths/min, open mouth breathing and short, rapid, shallow breaths). Due to the significant side effects, the 30 mg/kg dose is discontinued and for the subsequent dosing days, cats of this group are dosed at 5 mg/kg. At this lower dose, the only untoward effect noted is mydriasis in both cats following each daily dose.

[0076] The two additional Group 4 cats dosed only once with bicifadine at 5 mg/kg for PK sample collection show mild and moderate mydriasis. The female cat vomits 17 min after dosing; demonstrates moderate hypersalivation; elevated respiratory rate until 2 h post dose; and mydriasis (unresponsive to papillary light during the first 2 h post dosing) is present until 24 h post dose. The male cat shows mild mydriasis and increased respiration until 2 h post dose.

[0077] Cats receiving a dose of 10 mg/kg bicifadine show on the first day mild and moderate mydriasis with poor pupillary light reflex. The female cat appears hyperaesthetic; shows moderate hypersalivation; and exhibits increased heart and respiratory rates until 2 h post dose. The male shows mydriasis and increased respiration. Clinical signs resolve by 2 h post dose and by 4 h post dose both cats appear normal other than mydriasis, which is sustained until 6 h for the female cat. The female cat shows similar clinical signs after the second dose - hyperaesthesia, moderate hypersalivation, increased respiratory rate until 2 h post dose - while the male shows mild mydriasis. After the third and fourth doses only mydriasis is observed in both cats.

[0078] Cats receiving a dose of 1 mg/kg bicifadine show on the first day mild mydriasis but the pupillary light reflex is present. Slightly increased respiration (60 breaths/min) is observed in the female cat, but it is not clear if this is dose related or due to the stress from serial blood collection.

[0079] Clinical responses in general are more pronounced following the first dose and the severity is dose dependent. The majority of the adverse events occur between 30 min and 2 h after dosing. More adverse events with higher severity are observed in female cats suggesting the possibility of a gender difference.

[0080] Clinical pathology data during acclimation, clinical chemistry data 4 h post dose on day 9, hematology and clinical chemistry 24 h post dose and at the end of four repeat dose period do not reveal any findings of clinical relevance.

[0081] No significant body weight changes are observed.

[0082] No findings of clinical or toxicological relevance other than mydriasis are observed during physical examination at 24 h post dose and at the end of the study.

Results

Behavioral observations, hematology and clinical chemistry

1. There are no clinically significant changes in hematology or clinical chemistry at any dose.

2. No cats at any dose have serious adverse events or die.

3. There is no significant change in body weight of the cats in any of the dose groups, indicating apparent good health and appetite.

4. The behavioral effects of bicifadine on the first day are confounded by the blood draws in spite of pre-dosing simulated blood draws. Several cats become agitated during the blood draws.

5. Clinical responses are less pronounced following the first dose and the severity is dose dependent. The events are short-lasting in duration and the majority of the clinical responses occur between 30 min and 2 h after dosing. 6. The clinical responses are consistent with the sympathomimetic (proadrenergic) pharmacology of bicifadine.

7. Clinical responses that are observed at the first dose are generally not seen after the first dose with the exception of mydriasis.

8. No findings of clinical or toxicological relevance other than mydriasis are observed during physical examination at 24 h post dose and at the end of the study.

9. More clinical responses with higher severity are observed in female cats suggesting the possibility of a gender difference.

10. Cats receiving a dose of 1 mg/kg bicifadine show on the first day mild mydriasis; however, the clinical and toxicological relevance of this finding is questionable. The only clinical responses for the female cat are short-lived mydriasis on all 4 treatment days. The clinical response for the male cat is short-lived mydriasis on day 1 only.

11. For the 10 mg/kg oral dose, the female cat has mydriasis, hypersalivation, hyperaesthesia, increased respiration and heart rate, and vomits in the pen on the first day. She has

hyperaesthesia and mydriasis on the second day and only short-lived mydriasis on the remaining two days. The male has increased respiration and mydriasis on day one, and short-lived mydriasis on the remaining 3 days.

12. Cats receiving a dose of 30 mg/kg bicifadine show marked clinical responses including pronounced mydriasis with poor or lack of pupillary light reflex; hyperaesthesia; moderate hypersalivation (female cat only); increased heart rate; and respiratory distress. The veterinarians conducting the study recommend that this dose be discontinued.

13. Due to the significant clinical responses, the 30 mg/kg dose is discontinued and for the subsequent dosing days, cats from this group are dosed at 5 mg/kg po. At this lower dose, the only clinical response noted is short-lived mydriasis in both cats following each daily dose. Pharmacokinetics results

1. Bicifadine is rapidly absorbed with a T max of 0.25-2.5 hour, consistent with appearance of clinical responses (see Table 2, Figures 1 and 2).

2. The Ti/2 of bicifadine varies from 2.5-9.2 hours depending on the dose, and consistent with the duration of clinical responses (Table 2, Figures 1 and 2).

3. Bicifadine has high plasma levels that increase with dose.

4. There is no apparent gender difference on plasma levels with bicifadine. 5. The plasma levels of a major metabolite, the lactam, are approximately equal to the plasma levels of bicifadine, and last longer than parent (Figure 3). The bicifadine lactam metabolite is shown below:

Table 2. Average pharmacokinetic parameters for bicifadine in cats

Table 3. Average pharmacokinetic parameters for bicifadine lactam metabolite in cats

Summary

[0083] Dose dependent adverse events are observed following oral administration of bicifadine at 1, 5, 10, and 30 mg/kg in cats. Clinical responses in general are more pronounced following the first dose and the severity is dose dependent. The majority of the adverse events occur between 30 min and 2 h after dosing. More adverse events with higher severity are observed in female cats suggesting the possibility of a gender difference.

[0084] Cats receiving a dose of 30 mg/kg bicifadine show moderate signs of toxicity including mydriasis with poor or lack of pupillary light reflex, changes in behavior, hypersalivation, increased heart rate, and respiratory distress.

[0085] Similar but less severe signs of toxicity are observed in cats receiving a dose of 10 mg/kg bicifadine following the first dose, and are more pronounced in the female cat. [0086] Cats receiving a dose of 5 mg/kg bicifadine show mild to moderate mydriasis. The female cat of the pair vomits; exhibits moderate hypersalivation after dosing; and has elevated respiratory rate after the first dose. Mydriasis is the only adverse event after subsequent doses.

[0087] Cats receiving a dose of 1 mg/kg bicifadine show on the first day mild mydriasis, however, the clinical and toxicological relevance of this finding is questionable. Most likely the mydriasis is due to the sympathomimetic effects of bicifadine.

Example 2

[0088] l-p-tolyl-3-azabicyclo[3.1.0]hexane hydrochloride is dissolved in a vehicle which consists of 1 part polyethylene glycol 400 and 19 parts of 2% aqueous starch suspension with a drop of Tween ® 80 added, unless noted otherwise.

[0089] Dogs are female beagles and weigh 8-12 kg. Dogs are fed approximately 3 hours before oral administration of drugs. Dogs are dosed by gavage using a No. 22 Fr. catheter.

Administration to Dogs - Acute

[0090] l-p-tolyl-3-azabicyclo[3.1.0]hexane hydrochloride is administered in 20 ml 2% starch by gavage, followed by 20 ml tap water to two female beagles. Dogs are removed from food at least two hours prior to dosing. Dogs are observed continuously for four hours, and at 24 hours post- treatment.

Administration to Dogs - Sub-Chronic

[0091] Dogs are adult female beagles weighing 8.0-11.5 kg. Doses of 25 and 50 mg/kg of 1-p- tolyl-3-azabicyclo[3.1.0]hexane hydrochloride are administered orally by gavage in 20 ml vehicle followed by 20 ml tap water. Dogs are removed from food at least 2 hours prior to dosing. Treatment is administered on three consecutive days. Dogs are observed for effects for at least 4 hours and again on the following day before treatment. Dogs are also observed on subsequent days after dosing until effects subside.

Results

Administration to Dogs - Acute

[0092] l-p-tolyl-3-azabicyclo[3.1.0]hexane hydrochloride administered orally at a dose of 50 mg/kg to two female beagles causes decreased activity, curiosity, responsiveness, and respiration rate; impairment or loss of pupillary constriction response to light, mydriasis, occasional slight twitches, ataxia, and exophthalmos. The onset of effect is less than one hour, the duration of treatment is between 6 and 18 hours, and the time of peak effect is two hours post treatment. Administration to Dogs - Sub-Chronic

[0093] The major signs after oral administration of 25 and 50 mg/kg are decreased activity, mydriasis, impairment, or loss of pupillary constriction response to light, salivation, hindlimb rigidity, and loss of body weight. The effects are exacerbated with each successive treatment, and mydriasis and rigidity persist for up to four days after final treatment. Due to anorexigenic effects of repeat dosing with l-p-tolyl-3-azabicyclo[3.1.0]hexane hydrochloride, dogs are fed with canned dog food rather than regular laboratory chow. Body weights gradually return to pretreatment values in about 2 weeks.

Example 3

Summary and Conclusions

[0094] Administration of l-p-tolyl-3-azabicyclo[3.1.0]hexane hydrochloride at 35 mg/kg, p.o. to conscious, trained, normotensive dogs is without significant effect on arterial blood pressure, heart rate, all standard limb leads of the electrocardiogram, and the response of the tyramine challenge at 2 and 4 hours post dosing. However, at this high dose, all of the dogs exhibit hyperesthesia, mydriasis, vasodilation of the skin, and rigidness of the limbs.

Methods

[0095] Female beagle dogs, which weigh from 6.6-10.0 kg, are trained to lie quietly in a supine position with the four limbs restrained with leather straps. Arterial blood pressure is monitored by puncturing the femoral artery with a 26G 1½" thin wall needle attached to a Statham P23 Db transducer. All standard limb leads (I, II, III, AVR, AVL, AVF) of the electrocardiogram are recorded, and the heart rate is obtained by electronic integration of the ECG.

[0096] The tyramine challenge consists of a dose of 200 g/kg injected into the left cephalic vein. l-p-tolyl-3-azabicyclo[3.1.0]hexane hydrochloride, 35 mg/kg is dissolved in 20 cc of universal vehicle, or 20 cc of universal vehicle (serving as control), is given by gavage via a stomach tube. The universal vehicle consists of: 1 cc PEG 400, 2 drops of Tween 80, and 18.8 cc of 2% arrowroot starch. The stomach tube is flushed with 20 cc of 0.85% saline.

[0097] The cardiovascular parameters and the tyramine challenge are recorded prior to and at 2 and 4 hours after the administration of drug or the universal vehicle. Each dog serves as its own control. l-p-tolyl-3-azabicyclo[3.1.0]hexane hydrochloride and universal vehicle are tested in parallel in each experiment, and the dogs are randomly assigned to either group.

Statistical Analysis

[0098] The results from the above described experiments are analyzed for statistical significance using dependent and independent "t" tests for data from within and between groups respectively. Results

[0099] Neither l-p-tolyl-3-azabicyclo[3.1.0]hexane hydrochloride at 35 mg/kg dissolved in 20 cc of universal vehicle, nor 20 cc of the universal vehicle (serving as control), administered to conscious normotensive dogs via gavage, produce any statistically significant effects on the arterial blood pressure, heart rate, electrocardiogram, or tyramine challenge at 2 and 4 hours post dosing. There is no significant difference in the duration of the tyramine response after the administration of l-p-tolyl-3-azabicyclo[3.1.0]hexane hydrochloride or the universal vehicle (Table 4).

[00100] The following overt effects are noted 2 hours after the administration of 1-p-tolyl-

3-azabicyclo[3.1.0]hexane hydrochloride: hyperesthesia (4/4 dogs), mydriasis (4/4), vasodilation of the skin (4/4), rigidness of the limbs (4/4), salivation (2/4), tachypnea (2/4), diarrhea (1/4), and aggression (1/4).

Table 4. Effects of l-p-tolyl-3-azabicyclo[3.1.0]hexane hydrochloride on Mean Arterial Blood Pressure (MABP), Heart Rate (HR), and Tyramine Challenge in Conscious

Normotensive Dogs

MABP = mean arterial blood pressure in mm Hg

HR = heart rate in beats per minute

a = Changes in mean arterial blood pressure (in mm. Hg) from the control b = Mean value ± standard error

c = Statistically significant at p < 0.05 (independent "t" test) as compared with dogs treated with universal vehicle (control)

Example 4

[00101] l-p-tolyl-3-azabicyclo[3.1.0]hexane hydrochloride is administered orally (by capsule) in the morning once daily to four dogs (two male, two female) for 6 days at doses of 10- 60 mg/kg/day (calculated as the salt) (10 mg/kg on day 1 with succeeding doses increasing daily by 10 mg/kg).

[00102] One female dog is found dead approximately 23 hours after receiving 60 mg/kg on day 6 of the study. Signs of toxicity observed on day 6 include mydriasis, salivation, sedation, emesis, ataxia, and head drop.

[00103] Signs of toxicity in dogs that receive 10-60 mg/kg/day include slight loss of body weight, anorexia, mydriasis, loss of pupillary constriction response to light, emesis, sedation, salivation, lacrimation, ataxia, stiff hind legs and neck, head drop, muscle tremors, vocalization, rapid respiration, and conjunctival congestion. Mydriasis, salivation, lacrimation, sedation, ataxia, and stiff hind legs are noted throughout the period of drug administration but neither the incidence nor intensity of toxic signs appear to be related to dose. The onset of the signs range from 15-60 minutes and, in general, subside within 6 hours except for mydriasis, salivation, and lacrimation of slight intensity which is observed sporadically up to 6 hours after drug

administration.

Experimental Design

Animals

[00104] Four beagle dogs (2 male, 2 female) 15 months old at the start of drug

administration are used. They range in weight from 7.0-9.2 kg for males and 8.9-9.0 kg for females. The dogs are caged individually in a windowless, air conditioned room which is maintained at a temperature of 70-75° F and artificially illuminated for 12 hours (5:30 a.m. -5:30 p.m.) each day. The dogs are vaccinated against distemper, rabies, leptospirosis, parainfluenza, infectious canine hepatitis, and oral papilloma. They are offered 250 g (females) or 300 g (males) of food once daily approximately 1 hour after drug administration. The amount of food eaten is recorded after being offered and any food remaining is discarded. If food intake is markedly decreased, the food is allowed to remain in the cages overnight. Water is available at all times. Drug Administration

[00105] Drug is administered orally (by capsule) once daily in the morning. The first dose is 10 mg/kg with succeeding doses increasing daily by 10 mg/kg. Capsules are prepared daily prior to each dose and are based on body weights obtained predose and on day 4.

Methods

Observations

[00106] The dogs are observed continuously for signs of toxicity for approximately 2 hours after drug administration and then at half hour intervals for the next 5-6 hours each day.

Food intake is recorded daily and body weights are obtained predose and day 4.

Results

Survival

[00107] One female dog is found dead approximately 23 hours after receiving a dose of 60 mg/kg (day 6). The dog previously receives 5 daily doses beginning with 10 mg/kg on day 1 which is then increased by 10 mg/kg each succeeding day. Signs of toxicity observed on day 6 include emesis, mydriasis, salivation, sedation, ataxia, head drop, and stiff hind legs. The dog has a slight loss of body weight and a marked decrease in food intake. In postmortem findings, there are no gross or light microscopic findings that can account for the death. There are no

morphologic changes associated with mortality.

Food Intake and Body Weight

[00108] There is a slight loss of body weight in two of the three dogs that survive the period of administration. Food intake in the surviving three dogs is decreased in spite of the food being allowed to remain in the cages overnight on days 4-5 of the study.

Observations

[00109] Observations are shown in Table 5. Table 5. Observations

a PCR = pupillary constriction response to light

[00110] Signs of toxicity observed include mydriasis, loss of pupillary constriction response to light, emesis, sedation, salivation, lacrimation, ataxia, stiff hind legs and neck, head drop, muscle tremors, vocalization, rapid respiration, and conjunctival congestion. Mydriasis, salivation, lacrimation, sedation, ataxia, and stiff hind legs are noted throughout the period of drug administration. The onset of the signs observed range from 15-60 minutes after drug administration and, in general, subside within 6 hours except for slight mydriasis and occasional slight salivation or lacrimation which is observed sporadically up to 6 hours after drug administration. Neither the incidence nor the intensity of the above signs appear to be related to the dose.