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
  • A61K31/16—Amides, e.g. hydroxamic acids
  • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/04—Centrally acting analgesics, e.g. opioids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C233/00—Carboxylic acid amides
  • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
  • C07C233/16—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms
  • C07C233/17—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
  • C07C233/22—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to an acyclic carbon atom of a carbon skeleton containing six-membered aromatic rings

Definitions

• the present invention relates to isolated stereoisomeric forms of the compound (S)2-N(3- 0-(propan 2-ol)-l-propyl-4-hydroxybenzene)-3-phenylpropylamide. Specifically, the present invention relates to the use of (S)2-N(3-0-((S)propan 2-ol)-l-propyl-4- hydroxybenzene)-3-phenylpropylamide in the treatment or prophylaxis of pain, in particular, neuropathic pain.
• the compound 2-N(3-0-(propan 2-ol)-l-propyl-4-hydroxybenzene)-3-phenylpropylamide is disclosed in U.S. 7,754,771, and has proven to be efficient in the treatment of HIV-1 infection.
• This compound has been further disclosed for use in the treatment or prophylaxis of pain and inflammation in WO 2009/1099850, WO 2011/030105 and US 2011/0086910.
• Previous disclosures on this compound have related to the racemate containing all four enantiomers and diastereomers, namely (S,S), (S,R), (R,R) and (R,S).
• the racemate containing the S enantiomers at the chiral position adjacent to the amide were disclosed as a particularly advantageous embodiment. Pain is a multifaceted or multidimensional, experiential response to a variety of stimulus conditions. Pain is defined by the International Association for the Study of Pain (IASP) as "an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage
• Pain can be acute or chronic. Acute pain is usually caused by soft tissue damage, infection and/or inflammation among other causes. Chronic pain may have no apparent cause or may be caused by a developing illness or imbalance. Chronic pain is defined as the disease of pain; its origin, duration, intensity and specific symptoms may vary. Moreover, chronic pain can be classified as either nociceptive or neuropathic. Nociceptive pain includes tissue injury-induced pain and inflammatory pain such as that associated with arthritis.
• Nociceptive pain has been traditionally managed by administering non-opioid analgesics, such as acetylsalicylic acid, choline magnesium trisalicylate, acetaminophen, ibuprofen, fenoprofen, difhisinal, and naproxen; or opioid analgesics, including morphine, hydromorphone, methadone, levorphanol, fentanyl, oxycodone, and oxymorphone.
• Neuropathic pain a debilitating chronic pain following nerve damage, is characterized by its chronic nature, hyperalgesia, or abnormal pain hypersensitivity to innocuous stimuli (tactile allodynia).
• Hyperalgesia is an exaggerated response to a painful stimulus.
• AUodynia is the perception of normal stimuli as painful (examples include the touch of clothing, warm or cool air, etc.).
• Neuropathic pain can be a sequel to nerve damage in an extremity such as an arm, or more often, a leg. Precipitating events can include trauma or amputations (e.g., phantom limb pain).
• Neuropathic pain can occur due to an adverse effect of drug therapies, e.g., vincristine or paclitaxel (TaxolTM), or can occur as a component of disease pathologies, such as diabetes type 1 or type 2, shingles, HIV-1 infections, etc.
• neuropathic pain does not respond to opiates or non-steroidal anti-inflammatory drugs such as aspirin.
• Current suggested medicaments to Neuropathic pain include anti- epileptics (e.g., gabapentin, carbamazepine, valproic acid, topiramate, phenyloin), NMDA antagonists (e.g., ketamine, dextromethorphan), topical lidocaine (for post-herpetic neuralgia), and tricyclic antidepressants (e.g., fluoxetine, sertraline and ami trip tyline).
• anti- epileptics e.g., gabapentin, carbamazepine, valproic acid, topiramate, phenyloin
• NMDA antagonists e.g., ketamine, dextromethorphan
• topical lidocaine for post-herpetic neuralgia
• tricyclic antidepressants e.g.,
• Lyn tyrosine kinase a member of the Src family kinases (SFKs) plays an important role in the pathogenesis of neuropathic pain. Lyn expression in the spinal cord was highly restricted to microglia, and its level was increased after nerve injury. It has been shown that mice lacking Lyn exhibit a striking reduction in their ability to tactile allodynia after nerve injury. It was concluded that neuropathic pain may be linked to up-regulation of Lyn tyrosine kinase. (See Tsuda et al., Glia, 2008, 56:50-8).
• the present invention relates to isolated stereoisomeric forms of the compound (S)2- 0-(propan 2-ol)-l-propyl-4-hydroxybenzene)-3-phenylpropylamide of formula I.
• the present invention relates to the isolated enantiomer (S)2-N(3-0-((S)propan 2-ol)-l-propyl-4-hydroxybenzene)-3-phenylpropylamide) for use in the treatment or prophylaxis of pain, in particular neuropathic pain.
• the present invention is based in part on the surprising discovery that the substantially pure enantiomers (S)2-N(3-0-((S)propan 2-ol)-l-propyl-4-hydroxybenzene)-3- phenylpropylamide (also known as the (S,S) enantiomer or El) and (S)2-N(3-0- ((R)propan 2-ol)-l -propyl -4-hydroxybenzene)-3-phenylpropyl amide (also known as the (S,R) enantiomer or E2) modulate the activity of specific tyrosine kinases in an opposite manner.
• the present invention provides a compound of formula II
• the present invention provides a compound of formula III
• substantially pure when used to describe an enantiomer of a compound means that one enantiomer is substantially free of other stereoisomeric forms of the compound.
• a representative substantially pure enantiomer comprises greater than 90% by weight of one enantiomer of the compound and less than 10% by weight of the other stereoisomeric forms of the compound, preferably greater than 95% by weight of one enantiomer of the compound and less than 5% by weight of the other stereoisomeric forms of the compound, even more preferably greater than 98% by weight of one enantiomeric form of the compound and less than 2% by weight of the other stereoisomer ⁇ forms of the compound.
• other stereoisomeric forms may include at least one of the (S,S), (S,R), (R,S) and (R,R) enantiomeric or diastereomeric forms of the compound 2-N(3-0-(propan 2-ol)-l-propyl-4- hydroxybenzene)- 3 -phenylpropylamide .
• the substantially pure enantiomer of formula II ((S,S) enantiomer) is capable of activating BLK and LynA tyrosine kinases.
• the substantially pure enantiomer of formula III (S,R enantiomer) is capable of inhibiting BLK and LynA tyrosine kinases.
• the (S,S) enantiomer had no effect on the activity of LynB tyrosine kinase while the (S,R) enantiomer inhibited its activity.
• the present invention provides a therapeutic agent for the treatment of pain, comprising an activator of BLK and/or LynA tyrosine kinases as the active ingredient.
• the pain is an acute pain.
• the pain is a chronic pain.
• the chronic pain is nociceptive pain.
• the chronic pain is neuropathic pain.
• the activator of BLK and/or Lyn A tyrosine kinase is a substantially pure (S)2-N(3-0-((S)propan 2-ol)-l-propyl-4-hydroxybenzene)-3- phenylpropylamide or a pharmaceutically acceptable salt or hydrate thereof.
• the neuropathic pain is at least one symptom selected from neuropathic pains in post herpetic neuralgia, trigeminal neuralgia, diabetic neuralgia, cancer pain, persistent postoperative or post-traumatic pain, hyperalgia, allodynia, post- thoracotomy pain, CRPS, pain associated with multiple sclerosis, AIDS, thalamic pain, paraplegic pain caused by myelopathy, anesthesia dolorosa and phantom limb pain.
• the present invention provides a pharmaceutical composition comprising an activator of BLK and/or LynA tyrosine kinases as an active ingredient.
• the pharmaceutical composition comprises a substantially pure (S)2-N(3-0- ((S)propan 2-ol)-l-propyl-4-hydroxybenzene)-3-phenylpropylamide or a pharmaceutically acceptable salt or hydrate thereof and a pharmaceutically acceptable carrier or diluent.
• the pharmaceutical composition is useful for the treatment of pain.
• the pain may be acute or chronic.
• the chronic pain may be nociceptive pain or neuropathic pain.
• the pharmaceutical composition is formulated as a unit dosage form.
• the unit dosage comprises from 0.1 to 500 mg of substantially pure (S)2-N(3-0-((S)propan 2-ol)-l-propyl-4-hydroxybenzene)-3- phenylpropylamide or a pharmaceutically acceptable salt or hydrate thereof.
• the pharmaceutical composition is formulated for oral administration.
• the present invention provides methods for the treatment or prophylaxis of pain in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising as an active ingredient a compound comprising substantially pure (S)2-N(3-0-((S)propan 2-ol)-l-propyl-4-hydroxybenzene)-3- phenylpropylamide or a pharmaceutically acceptable salt or hydrate thereof.
• the methods of the present invention comprise administering a daily dose of 1.0 mg to 15 g of the active ingredient.
• the methods of the invention provide analgesia over a period of 24 hours after a single administration of the pharmaceutical composition.
• the present invention further relates to the use of substantially pure (S)2-N(3-0-((S)propan 2-ol)-l-propyl-4-hydroxybenzene)-3-phenylpropylamide or a pharmaceutically acceptable salt or hydrate thereof for the preparation of a medicament for the treatment or prophylaxis of pain.
• the pain is neuropathic pain.
• the methods of the invention provide analgesia over a period of 24 hours after a single administration of the pharmaceutical composition.
• Figure 1 shows the mean latency time measures using the tail flick test.
• Study groups from left to right include: vehicle (group 1); Morphine (group 2); 3 mg/kg of substantially pure (S)2-N(3-0-((S)propan 2-ol)-l-propyl-4-hydroxybenzene)-3-phenylpropylamide (group 3); 10 mg/kg of substantially pure (S)2-N(3-0-((S)propan 2-ol)-l-propyl-4- hydroxybenzene)-3-phenylpropylamide (group 4); 20 mg/kg of substantially pure (S)2- N(3-0-((S)propan 2-ol)-l-propyl-4-hydroxybenzene)-3-phenylpropylamide (group 5) and 10 mg/kg of substantially pure (S)2-N(3-0-((R)propan 2-ol)-l-propyl-4-hydroxybenzene)- 3-phenylpropylamide (group 6).
• * p ⁇ 0.01 vs
• Figure 2 shows the difference between post-treatment and pre-treatment latency times measured using the tail flick (sec).
• Study groups from left to right include: vehicle (group 1); Morphine (group 2); 3 mg/kg of substantially pure (S)2-N(3-0-((S)propan 2-ol)-l- propyl-4-hydroxybenzene)-3-phenylpropylamide (group 3); 10 mg/kg of substantially pure (S)2-N(3-0-((S)propan 2-ol)-l-propyl-4-hydroxybenzene)-3-phenylpropylamide (group 4); 20 mg/kg of substantially pure (S)2-N(3-0-((S)propan 2-ol)-l-propyl-4- hydroxybenzene)-3-phenylpropylamide (group 5) and 10 mg/kg of substantially pure (S)2- N(3-0-((R)propan 2-ol)-l-propyl-4-hydroxybenzene)-3-phenylpropylamide (group 6).
• FIG. 3 shows the mean group body weight of the different study groups measured during the course of the study (day 1, 7, 14 and 21).
• Study groups from left to right include: vehicle (group 1); Gabapentin (group 2); 30 mg/kg of substantially pure (S)2-N(3-0- ((S)propan 2-ol)-l-propyl-4-hydroxybenzene)-3-phenylpropylamide (group 3); 15 mg/kg of substantially pure (S)2-N(3-0-((S)propan 2-ol)-l-propyl-4-hydroxybenzene)-3- phenylpropylamide (group 4); 7.5 mg/kg of substantially pure (S)2-N(3-0-((S)propan 2- ol)-l-propyl-4-hydroxybenzene)-3-phenylpropylamide (group 5).
• Figure 4 shows the mean Von Frey force required for withdrawal of left operated leg measured on the 14 th day of the study 2, 5 and 24 hours post treatment and on the 21 st day of the study 2, 5, 24 and 48 hours post 7 days treatment.
• Study groups from left to right include: vehicle (group 1); Gabapentin (group 2); 30 mg/kg of substantially pure (S)2-N(3- 0-((S)propan 2-ol)-l-propyl-4-hydroxybenzene)-3-phenylpropylamide (group 3); 15 mg/kg of substantially pure (S)2-N(3-0-((S)propan 2-ol)-l-propyl-4-hydroxybenzene)-3- phenylpropylamide (group 4); 7.5 mg/kg of substantially pure (S)2-N(3-0-((S)propan 2- ol)-l-propyl-4-hydroxybenzene)-3-phenylpropylamide (group 5).
• Figure 5 shows the difference (delta) force between right healthy leg and left operated leg measured on the 14 th day of the study 2, 5 and 24 hours post treatment and on the 21 st day of the study 2, 5, 24 and 48 hours post treatment.
• Study groups from left to right include: vehicle (group 1); Gabapentin (group 2); 30 mg/kg of substantially pure (S)2-N(3-0- ((S)propan 2-ol)-l-propyl-4-hydroxybenzene)-3-phenylpropylamide (group 3); 15 mg/kg of substantially pure (S)2-N(3-0-((S)propan 2-ol)-l-propyl-4-hydroxybenzene)-3- phenylpropylamide (group 4); 7.5 mg/kg of substantially pure (S)2-N(3-0-((S)propan 2- ol)-l-propyl-4-hydroxybenzene)-3-phenylpropylamide (group 5).
• Figure 6 A-D shows the modulation the protein tyrosine kinases, BLK, LynA, LynB and Src by the compounds of the invention, presented in the form of IC 50 and EC 50 curves.
• Figure 7 A-C shows the chiral separation of (S)2-N(3-0-((S)propan 2-ol)-l-propyl-4- hydroxybenzene)-3-phenylpropylamide and (S)2-N(3-0-((R)propan 2-ol)-l-propyl-4- hydroxybenzene)-3-phenylpropylamide using a supercritical fluid chromatography (SFC) in combination with chiral stationary phases.
• the present invention relates to isolated stereoisomeric forms of the compound (S)2-N(3- 0-(propan 2-ol)-l-propyl-4-hydroxybenzene)-3-phenylpropylamide and to uses thereof for the therapeutic modulation of kinase-mediated processes, and treatment of disease and disease symptoms, particularly those mediated by certain kinase enzymes.
• the present invention related to the use of substantially pure (S)2-N(3-0-((S)propan 2-ol)-l- propyl-4-hydroxybenzene)-3-phenylpropylamide in the treatment of pain, preferably neuropathic pain.
• substantially pure when used to describe an enantiomer of a compound means that one enantiomer is substantially free of other stereoisomeric forms of the compound.
• a representative substantially pure enantiomer comprises greater than about 80% by weight of one enantiomer of the compound and less than about 20% by weight of other stereoisomeric forms of the compound, preferably greater than about 85% by weight of one enantiomer of the compound and less than about 15% by weight of other stereoisomeric forms of the compound, preferably greater than about 90% by weight of one enantiomer of the compound and less than about 10% by weight of the other stereoisomeric forms of the compound, more preferably greater than about 95% by weight of one enantiomer of the compound and less than about 5% by weight of the other stereoisomeric forms of the compound, more preferably greater than about 96% by weight of one enantiomer of the compound and less than about 4% by weight of the other stereoisomeric forms of the compound, even more
• other stereoisomeric forms may include at least one of the (S,S), (S,R), (R,S) and (R,R) enantiomeric or diastereomeric forms of the compound 2-N(3-0-(propan 2-ol)-l-propyl-4- hydroxybenzene)- 3 -phenylpropylamide .
• the term "about” as used herein denotes at most + 10 % of the value indicated, preferably no more than + 5%.
• the isolated enantiomers according to some embodiments of the invention may be synthesized as a racemate by known in the art methods described for example in US 7,754,771, US 7,642,290, US 7,674,829 or US 2011/0086910.
• the racemate may be further separated by known in the art methods for the separation of chiral compounds.
• the enantiomers may be synthesized as a racemate (comprising (S)2-N(3-0-((S)propan 2-ol)-l-propyl-4-hydroxybenzene)-3- phenylpropylamide and (S)2-N(3-0-((R)propan 2-ol)-l-propyl-4-hydroxybenzene)-3- phenylpropylamide and be further separated by a supercritical fluid chromatography (SFC) in combination with chiral stationary phases.
• SFC supercritical fluid chromatography
• the (S,S) and (S,R) compounds may be separated on RegisPackTM column a polysaccharide coated chiral column (with a tris-(3,5-dimethylphenyl) carbamoyl cellulose selector) generally used for enantiomeric separations of a wide range of racemate classes ( Figure 7A-C).
• the enantiomers may be synthesized directly using for example, the process described in scheme 1 for the preparation of the (S,S) enantiomer.
• the (S,R) enantiomer may be synthesized accordingly, by reacting (S)-OBnTyrosinol with (R)-Mesylate to form (S,R)- Bis-Protected.
• racemization occurs specifically at the first reaction step which involves the conversion of methyl lactate (S or R enantiomer) into 2-benzyloxy methyl lactate (S or R enantiomer).
• benzyl bromide and methyl lactate are to be mixed together prior to their
• the occurrence of racemization may be reduced upon performing the reaction at a temperature lower than room temperature, preferably, lower than 10°C, preferably, lower than 5°C, preferably, lower than 2°C, more preferably at a temperature lower than -10°C, most preferably at a temperature of -15°C or lower.
• the least racemization i.e. ⁇ 1% racemization
• the present invention provides a therapeutic agent for treating or preventing pain, preferably neuropathic pain, comprising (S)2-N(3-0-((S)propan 2-ol)-l-propyl-4- hydroxybenzene)-3-phenylpropylamide as an active ingredient; a pharmaceutical composition for treating or preventing pain, preferably neuropathic pain, comprising a therapeutically effective amount of (S)2-N(3-0-((S)propan 2-ol)-l-propyl-4- hydroxybenzene)-3-phenylpropylamide and a pharmaceutically acceptable carrier and methods for treating or preventing pain, specifically neuropathic pain comprising the administration of a pharmaceutical composition comprising a therapeutically effective amount of S)2-N(3-0-((S)propan 2-ol)-l-propyl-4-hydroxybenzene)-3-phenylpropylamide.
• Non limiting examples of neuropathic pain include neuropathic pains in post herpetic neuralgia, trigeminal neuralgia, diabetic neuralgia, cancer pain, persistent postoperative or posttraumatic pain, non-specific lower back pain, hyperalgia, allodynia, sciatica, postthoracotomy pain, CRPS, pain associated with multiple sclerosis, AIDS (or HIV- related neuropathy), fibromyalgia, thalamic pain, paraplegic pain caused by myelopathy, anesthesia dolorosa, phantom limb pain and the like.
• a therapeutic agent for neuropathic pain according to the present invention is especially effective for treating hyperalgia and allodynia.
• neuropathic pain conditions include pain associated with normally non-painful sensations such as "pins and needles" (paraesthesias and dysesthesias), 12
• hypoalgesia increased sensitivity to touch (hyperesthesia), painful sensation following innocuous stimulation (dynamic, static, thermal or cold allodynia), increased sensitivity to noxious stimuli (thermal, cold, mechanical hyperalgesia), continuing pain sensation after removal of the stimulation (hyperpathia) or an absence of or deficit in selective sensory pathways (hypoalgesia).
• terapéuticaally effective amount is that amount of the substantially pure (S)2- N(3-0-((S)propan 2-ol)-l-propyl-4-hydroxybenzene)-3-phenylpropylamide which is sufficient to provide a beneficial effect to the subject to which the substantially pure (S)2- N(3-0-((S)propan 2-ol)-l-propyl-4-hydroxybenzene)-3-phenylpropylamide is administered. More specifically, a therapeutically effective amount means an amount of the substantially pure (S)2-N(3-0-((S)propan 2-ol)-l-propyl-4-hydroxybenzene)-3- phenylpropylamide effective to alleviate or ameliorate the symptoms of pain in the subject being treated.
• treatment refers to symptomatic treatment and the term “prophylaxis” is used to mean preventing symptoms in an already afflicted subject or preventing recurrence of symptoms in an afflicted subject and is not limited to complete prevention of an affliction.
• the therapeutic agent for the treatment or prevention of pain according to the present invention is selected from substantially pure (S)2-N(3-0-((S)propan 2-ol)-l-propyl-4- hydroxybenzene)-3-phenylpropylamide may be administered orally or parenterally with no specific limitation on the manner of administration.
• (S)2-N(3-0-((S)propan 2-ol)-l-propyl- 4-hydroxybenzene)-3-phenylpropylamide as an active ingredient of the therapeutic agent for neuropathic pain according to the present invention may be provided independently, or provided as being contained in a formulation together with a pharmaceutically acceptable carrier or a pharmaceutical additive.
• composition of the present invention may be formulated as a unit dosage form.
• Each unit dosage form may comprise all or a predetermined fraction of the daily 13
• dose amount of the one or more compounds of the invention e.g., one half or one quarter of the daily dose amount.
• the composition may be formulated as a tablet, a pill, a capsule, a powder, fine granule, granules, a sterile parenteral solution or suspension, a metered aerosol or liquid spray, syrup, drops, an ampoule, an auto-injector device, a suppository, a cream or a gel.
• Said composition may be adapted for oral, enteral, parenteral, intrathecal, intranasal, sublingual, rectal or topical administration, or for administration by inhalation or insufflation.
• Oral compositions such as tablets, pills, capsules or wafers are particularly preferred.
• said one or more compounds may be mixed with one or more pharmaceutical excipients, e.g., microcrystalline cellulose, sodium citrate, calcium carbonate, dipotassium phosphate, glycine and the like may be used together with any of various disintegrators such as starch (preferably corn, potato or tapioca starch), alginic acid, a certain type of silicate double salt and the like; and a granule-forming binder such as polyvinylpyrrolidone, sucrose, gelatin, gum arabic or the like.
• a lubricant such as magnesium stearate, sodium lauryl sulfate, talc or the like is often very effective for tablet formation.
• a gelatin capsule filled with the same type of solid composition may be used.
• Substances preferably usable in connection with this include lactose as well as high-molecular-weight polyethylene glycol.
• the active ingredient may be used together with any of various types of sweeteners, flavorings, coloring agents or dyes, optionally an emulsifier and/or a suspending agent, as well as a diluent such as water, ethanol, propylene glycerol, glycerol, or the like or a combination thereof.
• the solid pre-formulation composition is then subdivided into unit dosage forms of the kind mentioned above which each may contain from 0.1 mg to about 500 mg of the compounds of the invention.
• the unit dosage forms contain from 1 mg to 500 mg, e.g. 1, 5, 10, 25, 50, 100, 200, 300 or 500 mg of the compounds.
• said tablet or pill when formulated as a tablet or pill, may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action.
• said tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former.
• enteric layer that serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release.
• enteric layers or coatings such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.
• the pharmaceutical composition of the present invention may be formulated as a liquid dosage form for administration orally or parentally by injection suppository and the like; for example an aqueous solution, a suitably flavored syrup, an aqueous or oil suspension or a flavored emulsion with edible oils such, for example, as cottonseed oil, sesame oil, coconut oil or peanut oil, as well as an elixir or a similar pharmaceutical vehicle.
• Suitable dispersing or suspending agents for an aqueous suspension include synthetic and natural gums, e.g., tragacanth, acacia, alginate, dextran, sodium carboxymethylcellulose, methylcellulose, polyvinyl-pyrrolidone or gelatin.
• the aqueous solution is appropriately buffered (preferably to pH 8 or higher) to first isotonize the liquid diluent.
• Such an aqueous solution is suitable for intravenous injection, and an oleaginous solution is suitable for intraarticular injection, intramuscular injection and subcutaneous injection.
• the liquid dosage form can be easily produced under aseptic conditions by a standard pharmaceutical technology well known to those skilled in the art.
• the active ingredient of the present invention may be topically administered to skin or the like. In such a case, it is desirable to topically administer the active ingredient in the form of cream, jelly, paste or ointment in accordance with the standard pharmaceutical practice.
• a therapeutic agent for neuropathic pain according to the present invention may be administered in an appropriate dose, with no specific limitation, which is selected in accordance with various conditions including the type of pain, age or symptom of the patient, administration route, purpose of treatment, and presence or absence of another medication used together with the agent.
• a daily dose of the therapeutic agent for neuropathic pain according to the present invention is, for example, about 0.1 mg/Kg body weight to about 100 mg/Kg body weight, preferably 1 to 50 mg/Kg body weight, more preferably 5-30 mg/Kg body weight. 15
• step 2 (S)-2-benzyloxypropylene glycol was then reacted with methane sulfonyl chloride in methylene chloride in the presence of triethyl amine to get the mesylate in 88% yield.
• a solution of step 2 was stirred in methylene chloride and methane sulfonyl chloride was added to it dropwise at ⁇ 5°C. After the addition was completed, the progress of the reaction was monitored by the TLC system. The reaction was quenched with USP-PW water. After the layers were separated, the aqueous layer was back extracted with methylene chloride. The methylene chloride layers were then combined and washed with USP-PW water 3 times to remove most of the methane sulfonic acid. No racemization occurred in this step. 16
• step 4 340 mg of the product of step 4 was reduced by hydro genation in the presence of 10% palladium on carbon catalyst and hydrochloric acid using methylene chloride as a solvent at 50°C. The reaction went to completion in approximately 4 hours with no racemization to yield El in 84.3% yield and 98.9%.
• the E2 enantiomer was synthesized as the racemate of the (S,S) and the (S,R) enantiomers as described in US 2011/0086910 and was further separated on RegisPackTM column, a polysaccharide coated chiral column (with a tris-(3,5-dimethylphenyl) carbamoyl cellulose selector) (Figure 7A-C).
• Example 3 Evaluation of the analgesic effect of El and E2 in a tail flick test:
• Morphine Preparation 19 ml saline was added to 1 ml morphine ampoule and 0.1 ml of received solution was injected per 20 g mice (5 ml/kg).
• test system See Table 1: The experiment was performed using male ICR strain 7 weeks old mice (25-27 g) (supplied by Harlan Israel, Ltd.)
• the experiment included a total number of 18 mice (part A) and 20 mice (part B).
• Animals were acclimated for 5 days. During acclimation and throughout the entire study duration, animals housed within a limited access rodent facility and kept in groups with a maximum of 6 mice per polypropylene cages. The cages were fitted with solid bottoms and filled with sterile wood shavings as bedding material. Animals were provided ad libitum with a commercial, sterile rodent diet and had free access to drinking water that was supplied to each cage via polyethylene bottles with stainless steel sipper tubes. Water was monitored periodically. Automatically controlled environmental conditions were set to maintain temperature at 20-24°C with a relative humidity (RH) of 30-70%, a 12:12 hour light:dark cycle and 15-30 air changes/h in the study room. Temperature and RH were monitored daily.
• RH relative humidity
• Table 1 Test groups and dose regimen of the present study:
• the heat source and timer are turned on by a foot pedal press, and automatically switch off when the animal flicks its tail off the emitter. Latency is measured and analyzed as an analgesic effect.
• the baseline tail flick was measured a day before the experiment started. On the day of the experiment, animal body weights were first measured followed by the IP administration of the test items (vehicle, morphine, NRD 13 S El and E2). 30 minutes after administration of the test items, the animals were subjected to the tail-flick testing. The tail-flick experiments were followed by blood sampling of all groups. The experiment was terminated after spinal cords and brains of all groups have been collected. Statistics and data evaluation: All parameters are represented as means and standard error of the mean (SEM). Data was analyzed using a two tailed, unpaired T-test to compare each treatment group to the Vehicle treated animals. A two-tailed, paired T-test was used to compare the pre-treatment and post-treatment of each treatment group. Probability values p ⁇ 0.01 and p ⁇ 0.05 are considered significant. Results:
• Body Weight The mean body weight for all the animals was 27.42+0.22 g. No statistical differences were found between groups.
• the Latency Time to Tail Flick (sec) are summarized in Table 2, columns 4-5 :
• the mean latency time for the Vehicle treated animals (Group 1) at baseline (pre-treatment) was 8.72+0.65 sec.
• Thirty minutes post drug administration the mean latency time was 7.78+0.36 sec, which is not statistically different from baseline.
• Treatment with morphine (Group 2) at a dose of 5 mg/kg was significantly effective in increasing the latency time when compared to the pre-treatment value: 7.92+0.17 sec pretreatment vs. 15.38+1.47 sec post-treatment (p ⁇ 0.01).
• Treatment with El at a dose of 10 mg/kg (Group 4) was significantly effective in increasing the latency time when compared to the pre-treatment value: 8.16+0.39 sec pretreatment vs. 10.63+0.64 sec post-treatment (p ⁇ 0.01).
• treatment with El at a dose of 10 mg/kg and 20 mg/kg expressed pain relief activity when compared to the Vehicle treated animals (Group 1): 10.63+0.64 sec (Group 4) and 10.65+0.81 sec (Group 5) vs. 7.78+0.36 sec Vehicle (p ⁇ 0.01).
• the antinociceptive and analgesic activity of El in a spinal nerve ligation (SNL or Chung) model for neuropathic pain in rats was evaluated.
• Vehicle 1 ml DMSO (Sigma) was dissolved in 4 ml saline (Supplier: TEVA medical).
• Gabapentin exists as a powder. To achieve a dose of 150 mg/kg, 250 mg of gabapentin was dissolved in 5 ml saline (50 mg/ml).
• a rat weighing 200 g was injected with 0.6 ml of dissolved solution.
• test groups and dose regimen of the present study are summarized in Table 3.
• Species 50 SD young adult male rats, 225-260 g at study initiation (Harlan Laboratories, Israel. Ltd.)
• Body Weight Weight variation of animals at the time of treatment initiation did not exceed +20% of the mean weight.
• Animal Health The health status of the animals used in this study was examined upon their arrival. Only animals in good health were acclimatized to laboratory conditions and were used in the study.
• Food and Water Animals were provided ad libitum with a commercial, sterile rodent diet and had free access to drinking water that was supplied to each cage via polyethylene bottles with stainless steel sipper tubes. A feed lot analysis of the diet batch used in the study was included in the archives with the study data. Water was monitored periodically.
• Each dosing group was kept in separate cages to avoid cross-contamination which could occur through consumption of fecal matter. 2 or 3 animals were housed per cage.
• Termination At the end of the study, surviving animals were euthanized by C0 2 .
• Table 3 test groups and dose regimen of the present study:
• the Chung model is a reliable model for neuropathy pain that enables the measurement of the animal's pain threshold immediately after the animal awakes from surgery.
• Neuropathic Pain Induction While under anesthesia using ketamine/xylazine sodium and after the area was shaved, the rat was placed in a prone position and the left paraspinal muscles were separated from the spinous process at the L4-S2 levels. The L6 vertebral transverse process was carefully removed with a small rongeur to visually identify the L5- L6 spinal nerves. The left L5-L6 spinal nerves were cut. The muscle was then closed with 4-0 silk sutures and the skin was closed by a clamp. Following surgery, the rats were returned to the cage and remained under a heating lamp until they awoke.
• Inclusion/ Exclusion Criteria for Pre-Selection On post-operative day 14, animals were tested for mechanical allodynia using Von Frey methodology prior to being placed in their experimental groups. Only animals with a pain threshold of ⁇ 26 g for the operated leg were included in the study. In order to form homogenous treatment groups and to adhere to randomization, all the operated rats were grouped according to inclusion/exclusion criteria.
• Treatment Treatment Commencement: Animals were selected using the parameter of pain development (Von Frey testing) and then placed into their experimental groups.
• Gabapentin the positive control, was administered only on the testing days 14, 15 and 21.
• Body Weights Body weight was measured at regular intervals on study days -1 for baseline values and seven days after the surgery (study day 7). In addition, animals that demonstrated criterion for mechanical allodynia on study day 14 were also weighed after selection and grouping on study days 14 and 21.
• Pain Response Evaluation Pain response was evaluated using Von Frey test for mechanical allodynia.
• the Von Frey test for mechanical allodynia is based on applying short pulses of pressure that are not painful to a naive animal. In fact, in order to achieve paw withdrawal from a naive animal, the pressure applied is sometimes higher than 60 g. This often requires the researcher to apply enough pressure with the Von Frey filament to actually lift the paw of the naive animal. However, in disease conditions, the animals are sensitive to much lower pressure and experience pain as a result of a normally non-painful stimulus.
• the rats were placed in an enclosure and positioned on a metal mesh surface, but allowed to move freely.
• the rats' cabins were covered with red cellophane to diminish environmental disturbances.
• the test began after cessation of exploratory behavior.
• the set of Von Frey monofilaments provide an approximate logarithmic scale of actual force and a linear scale of perceived intensity as provided by the manufacturer of the Von Frey apparatus (Ugo Basil).
• the operating principle When the tip of a fiber of given length and diameter is pressed against the skin at right angles, the force of application increases as long as the researcher continues to advance the probe until the fiber bends. After the fiber bends, the probe continues to advance, causing the fiber to bend more, but without additional force being applied to the paw. 28
• Rodents exhibit a paw withdrawal reflex when its paw is unexpectedly touched.
• the Touch TestTM Sensory Evaluator can be used on the plantar surfaces of the rat's foot. The animal will indicate sensation by pulling back its paw. The minimal force needed to elevate the withdrawal reflex is designated / considered as the value of reference.
• Body Weight (Table 6; Figure 3): All animals gained weight during the study; however, there were no significant differences in weight gain between the groups. Baseline body weight for all animals was 243.76+1.21 g on study day -1. Body weights were also measured on study days 7, 14 and 21. At study day 7, the mean body weight for all groups was 258.12+1.71 g. At study day 14, the mean body weight for all groups was 286.34+1.93 g. At study day 21, the mean body weight was 302.34+2.29 g.
• Gabapentin 150 mg/kg IP (Group 2): When compared to the Vehicle control (Group 1), treatment with gabapentin on study day 14 inhibited allodynia 2 hours after its administration; but was not effective 5 hours after its administration.
• El 7.5 mg/kg IP (Group 5 ): When compared to the pretreatment value, treatment with El at a dose of 7.5 mg/kg on study day 14 inhibited allodynia 2, 5 and even 24 hours after its administration. When compared to the pretreatment value, treatment with El at a dose of 7.5 mg/kg on study day 21 inhibited allodynia 5 hours after its administration. Gabapentin 150 mg/kg IP (Group 2): When compared to the pretreatment value, treatment with gabapentin on study day 14 inhibited allodynia 2 hours after its administration; but was not effective 24 hours after its administration.
• Gabapentin 150 mg/kg IP (Group 2): Calculation of delta withdrawal force between the two legs on day 14 and on day 21 showed significant pain relief activity for gabapentin at 2 hours after its administration
• treatment with gabapentin inhibited allodynia 2 hours after its administration however was not effective 5 hours post administration.
• treatment with gabapentin effectively inhibited allodynia 2 hours after its administration.
• the El at a dose of 30 mg/kg was effective as a pain analgesic item in the spinal nerve ligation model for neuropathic pain in rats as reflected in the parameters of mechanical allodynia at 5 post Test Item administration on study day 14, and at 2, 5 and 24 hours post Test Item administration on study day 21.
• El at a dose of 7.5 mg/kg was effective as a pain analgesic item as reflected in the parameters of mechanical allodynia at 2, 5 and 24 hours post Test Item administration on study day 14, and at 2, 5, 24 and 48 hours post Test Item administration on study day 21.
• Gabapentin the positive control in this study, was active at 2 hours after its administration on study days 14 and 21. In contrast to Test Items, the analgesic activity of gabapentin was no longer detected 5 hours, 24 hours and 48 hours post dosing.
• LabChip assays are separations-based, meaning that the product and substrate are electrophoretically separated, thereby minimizing interferences and yielding the highest data quality available on any screening platform.
• Z' factors for both the EZ Reader and LC3000 enzymatic assays are routinely in the 0.8 to 0.9 range.
• the advantages of the LabChip assays include high Z' values, few false positives, few false negatives and analytical quality reproducibility.
• the off-chip incubation mobility- shift kinase assay uses a microfluidic chip to measure the conversion of a fluorescent peptide substrate to a phosphorylated product.
• the reaction mixture from a microliter plate well, is introduced through a capillary sipper onto the chip, where the nonphosphorylated substrate and phosphorylated product are separated by electrophoresis and detected via laser-induced fluorescence.
• the signature of the fluorescence signal over time reveals the extent of the reaction.
• Assay Control Data All assays must pass internal QA/QC standards including a reference IC50 for each assay run which must be within one half log unit (plus or minus) of the accepted historical average. Assay control data are summarized in Table 9:

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