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WO2005041886A2 - Procedes permettant de generer ou d'augmenter des revenus lies au commerce des inhibiteurs de la douleur - Google Patents

Procedes permettant de generer ou d'augmenter des revenus lies au commerce des inhibiteurs de la douleur Download PDF

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Publication number
WO2005041886A2
WO2005041886A2 PCT/US2004/036186 US2004036186W WO2005041886A2 WO 2005041886 A2 WO2005041886 A2 WO 2005041886A2 US 2004036186 W US2004036186 W US 2004036186W WO 2005041886 A2 WO2005041886 A2 WO 2005041886A2
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alkyl
aryl
acid
group
inhibitor
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WO2005041886A3 (fr
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Daniela Salvemini
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Metaphore Pharmaceuticals Inc
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Metaphore Pharmaceuticals Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group

Definitions

  • the present invention relates generally to methods of doing business. More specifically, the present invention relates to methods of generating and/or increasing the revenue derived from pain inhibitors.
  • NSAIDs nonsteroidal analgesic/anti-inflammatory drugs
  • opioids opioids
  • NSAIDs operate by inhibiting cyclooxygenase enzymes and thereby the synthesis of prostaglandins.
  • Prostaglandins sensitize pain receptors which lower the pain threshold perceived by subjects making normal stimuli, such as touch and stretch sensations, painful.
  • NSAIDs can be quite effective at returning the lowered pain threshold to normal but do not elevate the pain threshold.
  • NSAIDs available over-the-counter include: ibuprofen (Advil®), naproxen (Aleve® or Naprosyn®), and aspirin (Bayer®).
  • Prescription NSAIDs include: celecoxib -Celebrex®, diclofenac - Voltaren®, etodolac - Lodine®, fenoprofen - Nalfon®, indomethacin - Indocin®, ketoprofen - Orudis®, Oruvail®, ketoralac -Toradol®, oxaprozin - Daypro®, nabumetone - Relafen®, sulindac - Clinoril®, tolmetin - Tolectin®, and rofecoxib - Vioxx®.
  • a second class of pain relievers operate by mimicking natural peptides such as enkephalins and endorphins to stimulate one or more of the ⁇ -, ⁇ - and /c-receptor systems in the nervous system.
  • Opioids elevate the pain threshold so that normally painful stimuli are perceived as less painful or even euphoric.
  • Opioids are commonly used in the clinical management of severe pain, including chronic severe pain of the kind experienced by cancer patients.
  • Each of these classes of compounds has inherent problems and limitations.
  • NSAIDs that are nonselective for the cyclooxygenase 2 (COX-2) produced in inflammation also inhibit constitutive cyclooxygenase 1 (COX-1 ), causing undesirable damage to the gastric mucosa.
  • opioids have limited effectiveness as analgesics in lowering an elevated threshold to normal and are generally used for mild to moderate pain. They are also ineffective drugs for elevation of the pain threshold above normal levels, which prevents their use in pain such as surgical pain where an underlying pathological condition has not elevated the pain threshold.
  • Opioid analgesics are antagonized by analogous N-allyl compounds such as naloxone.
  • opioids have problems with tolerance and dependency, so that over a course of therapy increasing dosages of compound are required to achieve the same level of analgesia, and cessation of opioid administration when analgesia is no longer needed elicits a withdrawal syndrome with unpleasant and potentially serious symptoms.
  • NASH Narcotic induced hyperalgesia
  • NMDA N-methyl-D-aspartate
  • NOS nitric oxide synthase
  • Non- selective NOS inhibitors such as NG-nitroarginine prevent and reverse morphine tolerance.
  • compositions, composition combinations and methods to treat pain by administering superoxide-mediated nitration inhibitors to a subject in need thereof may be administered to a subject in combination with a known nitration, pain or inflammation inhibitor to enhance the effect of such known inhibitors to extend the potency and efficacy of the pain inhibitor.
  • This invention provides a method for providing a service comprising the acts of identifying a nitration inhibitor effective to treat, prevent, reverse or inhibit pain in a subject; and preparing a therapeutically effective dosage form of said nitration inhibitor or pro-drug thereof.
  • a method for providing a service comprising the acts of identifying a combination of a nitration inhibitor or pro-drug thereof and a pain inhibitor or pro-drug thereof, said nitration inhibitor being effective to treat, prevent, reverse or inhibit pain in combination with said pain inhibitor in a subject, and such that said combination enhances an analgesic effect of the pain inhibitor in the subject; and preparing a therapeutically effective dosage form of the combination.
  • a method for generating revenue comprising the acts of identifying a nitration inhibitor effective to treat, prevent, reverse or inhibit pain in a subject; and preparing a therapeutically effective dosage form of said nitration inhibitor or pro-drug thereof.
  • a method for generating revenue comprising the acts of identifying a combination of a nitration inhibitor or pro-drug thereof and a pain inhibitor or pro-drug thereof, said nitration inhibitor being effective to treat, prevent, reverse or inhibit pain in combination with said pain inhibitor in a subject, and such that said combination enhances an analgesic effect of the pain inhibitor in the subject; and preparing a therapeutically effective dosage form of the combination.
  • a method for providing a service comprising the acts of identifying a nitration inhibitor effective to reverse opioid tolerance in a subject; and preparing a therapeutically effective dosage form of said nitration inhibitor or pro-drug thereof.
  • the relevant methods above may further comprise marketing the nitration inhibitor or pro-drug thereof in commerce.
  • the relevant methods above may also further comprise selling the nitration inhibitor or pro-drug thereof in commerce.
  • the relevant methods may further comprise marketing the nitration inhibitor, or pro-drug thereof, and pain inhibitor, or pro-drug thereof, combination in commerce.
  • the relevant methods above may also further comprise selling the nitration inhibitor, or pro-drug thereof, and pain inhibitor, or pro-drug thereof, combination in commerce.
  • the nitration inhibitor is selected from the group consisting of a NOS inhibitor, NO generating system inhibitor, NO scavenger, and any combination thereof.
  • the NOS inhibitor is selected from the group consisting of an eNOS inhibitor, iNOS inhibitor, nNOS inhibitor, and any combination thereof.
  • the eNOS inhibitor is selected from the group consisting of NG-monomethyl-L-arginine (LNMMA), NG nitro-L-arginine (L- NA), NG-nitro-L-arginine methyl ester (L-NAME), and any combination thereof;
  • the iNOS inhibitor is selected from the group consisting of L-N5-(1-iminoethyl)-ornithine (L-NIL), aminoguanidine, 1400W (N-3-aminomethyl-benzylacetamidine), and any combination thereof;
  • the nNOS inhibitor is selected from the group consisting of 7-nitroindazole (7- Nl), 3-bromo-7-nitroindazole, 6-nitroindazole, Ng-propyl-L-arginine, and any combination thereof
  • the NO scavenger is selected from the group consisting of hemoglobin, PTIO, NPO, and any combination thereof.
  • the nitration is initiated in a central sensitization pathway of the subject.
  • the pain is initiated in a central sensitization pathway of the subject.
  • the opioid tolerance is initiated in a central sensitization pathway of the subject.
  • the pain inhibitor is an opioid.
  • the opioid is selected from the group consisting of morphine, oxycontin, oxycodone, codeine, fentanyl and any combination thereof.
  • the pain inhibitor is an NSAID.
  • the NSAID is selected from the group consisting of ibuprofen (Advil®), naproxen (Aleve® or Naprosyn®), aspirin (Bayer®), celecoxib (Celebrex®), diclofenac (Voltaren®), etodolac (Lodine®), fenoprofen (Nalfon®), indomethacin (Indocin®), ketoprofen (Orudis® or Oruvail®), ketoralac (Toradol®), oxaprozin (Daypro®), nabumetone (Relafen®), sulindac (Clinoril®), tolmetin (Tolectin®), and rofecoxib (Vioxx®).
  • the nitration inhibitor is selected from the group consisting of a SO generating system inhibitor, SO scavenger, and any combination thereof.
  • the SO generating system inhibitor is a xanthine oxidase inhibitor.
  • the SO scavenger is represented by the following formula: wherein (a) one or more of R, R', R 1 f R' 1 f R 2 , R' 2 , R 3 , R' 3 , R 4 , R' 4 , R 5 , R's, Re, R' ⁇ , ?, RV.
  • R 3 , R's, R 9 , and R' ⁇ are independently selected from the group consisting of hydrogen, and substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylcycloalkyl, cycloalkenylalkyl, alkylcycloalkyl, alkylcycloalkenyl, alkenylcycloalkyl, alkenylcycloalkenyl, heterocyclic, aryl, aralkyl, hyaluronic acid, polyethyleneglycol, -OR 10 , -NR10R11, -COR10.
  • R 10 and R- ⁇ are independently selected from the group consisting of hydrogen and alkyl groups, and a and b are integers independently selected from 1 to 6), and substituents attached to the ⁇ -carbon of ⁇ -amino acids; and (b) optionally, one or more of R or R' and R 1 or R' 1 , R 2 or R' 2 and R 3 or R' 3> R 4 or R' and R 5 or R' 5> R 6 or R ' 6 and R 7 or R' 7 , or R 8 or R' 8 and R 9 or R' 9 together with the carbon atoms to which they are attached independently form a substituted or unsubstituted nitrogen containing heterocycle having 2 to 20 carbon atoms, which may be an aromatic heterocycle in which case the hydrogen attached to the nitrogen which is both part of the
  • R 4 , R 5 and R' 5 , R 6 and R' 6 , R 7 and R' 7l R 8 and R 8 , and R 9 and R' 9 together with the carbon atom to which they are attached independently form a substituted or unsubstituted and saturated, partially saturated, or unsaturated cyclic or heterocyclic having 3 to 20 carbon atoms; and (e) optionally, one or more of R, R', R 1 f R' ⁇ , R 2) R' 2 , R 3 , R' 3 , R 4 , R 4 , R 5 , R's, R 6 , R' 6 , R 7 , R' 7 , R 8 , R's, R 9 , and R' 9 together with a different one of R, R', R., R' ⁇ , R 2 , R 2 , R 3 , R's, R 4 , R' 4l R 5 , R' 5 , R 6 , R' 6 , R 7 , RV, Rs, R's
  • U and V are saturated cycloalkyl heterocycles having 3 to 20 carbon atoms. More preferably, U and V are saturated cycloalkyl heterocycles having 4 to 10 carbon atoms. Still more preferably, U and V are trans-cyclohexanyl fused rings. In alternatives referring to a W moiety, W is preferably a substituted pyridino moiety. More preferably, U and V are trans-cyclohexanyl fused rings and W is a substituted pyridino moiety.
  • transition metal M is preferably selected from the group consisting of Mn, Fe, Ni and V.
  • the nitration inhibitor is selected from the group consisting of a peroxynitrite generating system inhibitor, peroxynitrite scavenger, and any combination thereof.
  • the peroxynitrite scavenger is represented by a formula selected from the group of formulas consisting of: Structure I
  • R 3 , R 6 , Rg and R 12 are independently selected from the group consisting of H, alkyl, alkenyl, CH 2 , COOH, phenyl, pyridyl, and N-alkylpyridyl, such that phenyl, pyridyl and N-alkylpyridyl are:
  • N-Alkylpyridyl which are attached at a carbon atom; and wherein Phenyl is optionally substituted by a substituent selected from the group consisting of a halogen, alkyl, aryl, benzyl, COOH, CONH 2 , S0 3 H, N0 2 , NH 2 , N(R) 3+ and NHCOR', wherein R is selected from the group consisting of hydrogen, alkyl, aryl and alkaryl, and R' is alkyl; wherein Pyridyl is optionally substituted by a substituent selected from the group consisting of a halogen, alkyl, aryl, benzyl, COOH, CONH 2 , S0 3 H, N0 2 , NH 2 , N(R) 3+ and NHCOR', wherein R and R' are as defined above; and wherein N-Alkylpyridyl is optionally substituted by a substituent selected from the group consisting of a
  • R' is CH or N
  • Re, R7, Rs, R9, R10, R11, R. 2 , R13, R ⁇ , R15, and R16 are independently selected from the group consisting of H, SO 3 H, COOH, N0 2 , NH 2 , and N- alkylamino; and
  • X, Y, Z, M and n are as defined above; Structure III
  • R-i, R 5 , R 9 , and R ⁇ 3 are independently selected from the group consisting of a direct bond and CH 2 ;
  • R 2 , R'2, R , R' , Re, R'e, Rs, R's, R.o, R'10, R12, R'12, R ⁇ , R ⁇ , R-i ⁇ , and R' ⁇ 6 are independently selected from the group consisting of H and alkyl;
  • R 3 , R , Ru, and R 15 are independently selected from the group consisting of H and alkyl; and
  • X, Y, Z, M and n are as defined above;
  • B wherein R 1 ; R 5 , R 8 , and R 12 are independently selected from the group consisting of a direct bond and CH 2 ;
  • R 2 , R' 2 , R 4 , R 4 , R 6 , R' 6 , R 7 , Rg, R'g, R.1, R'n, R13, R'i3, and R 14 are independently
  • R-i, R 4 , R 8 , and R 12 are independently selected from the group consisting of a direct bond and CH 2 ;
  • R 2 , R' 2 , R 3 , R 5 , R' 5 , R 7 , R 9 , R'g, Ru, R'n, R 13 , R' 13 and R 1 are independently selected from the group consisting of H and alkyl;
  • R-io is H or alkyl;
  • X, Y, Z, M and n are as defined above;
  • D wherein Ri, R 4 , R 7 and R ⁇ 0 are independently selected from the group consisting of a direct bond and CH 2 ;
  • R 2 , R' 2 , R 3 , R 5 , R' 5 , Re, Rs, R's, Rg, R11, R'n and R ⁇ 2 are independently selected from the group consisting of H and alkyl; and
  • X, Y, Z, M and n are as defined above;
  • Ri, R 4 , R 8 and Ru are independently selected from the group consisting of a direct bond and CH 2 ;
  • R 2 , R3, R'3, Rs, R's, R7, RV, Rg, R10, R'10, R12, R'12 and R 3 are independently selected from the group consisting of H and alkyl;
  • R 6 is hydrogen or alkyl;
  • X, Y, Z, M and n are as defined above;
  • R , R 7 and Rio are independently selected from the group consisting of H and alkyl;
  • R2, R3, R'3, Rs, R's, Re, Rs, Rg, R'g, R-i 1, R'11 and R 2 are independently selected from the group consisting of H and alkyl; and
  • X, Y, Z, M and n are as defined above;
  • Ri, R 3 , R 4 , and R 6 are independently selected from the group consisting of H and alkyl;
  • R 2 and R 5 are independently selected from the group consisting of H, alkyl, S0 3 H, N0 2 , NH 2 , halogen, COOH and N(R) 3+ wherein R is as defined above; and
  • X, Y, Z, M and n are as defined above;
  • R , R 2 , R 3 , and R are independently selected from the group consisting of H, alkyl, S0 3 H, N0 2 , NH 2 , halogen, COOH and N(R) 3+ wherein R is as defined above; and X, Y, Z, M and n are as defined above; and Structure IV
  • R' 5 , R 6 , R' 6 , R 7 and R' 7 are independently selected from the group consisting of H, alkyl, alkoxy, N0 2 , aryl, halogen, NH 2 and S0 3 H, further wherein R 6 , R'e, R 7 and R' 7 together with one other of R 6 , R'e, R and R' 7 optionally form a heterocycle having 5 to 8 carbon atoms and form a ring with the carbon atoms of the macrocycle to which they are attached; M 1 is selected from the group consisting of Fe, Ni or V; and X, Y, Z and n are as defined above.
  • a method wherein the nitration inhibitor is selected from the group consisting of a SO generating system inhibitor, SO scavenger, NOS inhibitor, NO generating system inhibitor, NO scavenger, peroxynitrite generating system inhibitor, peroxynitrite scavenger, and any combination thereof.
  • the pain inhibitor is an opioid or NSAID and the nitration inhibitor is selected from the group consisting of a SO generating system inhibitor, SO scavenger, NOS inhibitor, NO generating system inhibitor, NO scavenger, peroxynitrite generating system inhibitor, peroxynitrite scavenger, and any combination thereof.
  • the subject is a mammal.
  • the mammal is a human.
  • Figure 1 Structures of synthetic SODm M40403 and M40404.
  • Figure 2. Decay of superoxide (top line) uncatalyzed and in the presence of an active SOD mimetic (M40403, 5 ⁇ M; bottom line) and an inactive analog (M40404, 5 ⁇ M; middle line).
  • the active catalyst affords a pure 1st-order decay of superoxide while the uncatalyzed and M40404 decays remain pure 2nd-order decays.
  • Figure 3
  • Intrathecal injection of NMDA (2 nmol, ⁇ ) causes thermal hyperalgesia (when compared to vehicle, ⁇ ) and this response is blocked by subcutaneous injection of M40403 (10 mg/kg, given subcutaneously 30 min before NMDA, •).
  • Each point represents the mean ⁇ s.e.m. for 6 rats.
  • Figure 5 Inhibition of NMDA-induced hyperalgesia by M40403 is associated with inhibition of spinal manganese superoxide dismutase (MnSOD).
  • NMDA-induced hyperalgesia seen at time of peak nociception (20 minutes, B) is associated with nitration of the endogenous MnSOD at the level of the spinal cord (A).
  • Nitrated MnSOD is deactivated as shown by the reduced capacity of the enzyme to dismute superoxide (C, p ⁇ 0.05; unpaired Students t-test was employed as statistical analysis test for Fig. 5C).
  • M40403 (10 mg/kg, n 6) attenuates spinal MnSOD nitration (A) reducing the hyperalgesic response (B).
  • Immunoprecipitation data shown in panel A is representative of 6 experiments. Rat brain treated with peroxynitrite was used as positive control.
  • M40403 inhibits nitrotyrosine formation and poly-ADP-ribose- polymerase (PARP) activation in the carrageenan-inflamed rat hindpaw.
  • PARP poly-ADP-ribose- polymerase
  • SO is also formed and plays a major role in the development of pain through direct peripheral sensitization, through the release of various cytokines (TNF ⁇ , IL-1 ?, IL-6), through the formation of peroxynitrite (ONOO " ) and through PARP activation.
  • SO when released at the level of the spinal cord in response to presumably glutamate (see discussion) SO in turn interacts with NO to form ONOO " .
  • the latter in turn nitrates and subsequently deactivates MnSOD in the spinal cord.
  • levels of SO remain elevated favoring the maintenance of nociceptive signaling.
  • the anti-nociceptive effect of M40403 includes overall inhibition of the effects of SO exerted peripherally and centrally.
  • Controlled-Release Component refers to a composition or compound, including, but not limited- to, polymers, polymer matrices, gels, permeable membranes, liposomes, microspheres, or ' the like, or a combination thereof, that facilitates the controlled-release of a composition or composition combination.
  • Pharmaceutically Acceptable As used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S.
  • compositions for use in animals, and more particularly in humans.
  • pharmaceutically acceptable carrier refers to a diluent, adjuvant, excipient, or vehicle with which a composition is administered.
  • Such carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents. Water is a preferred carrier when a composition is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • a composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates.
  • Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for the adjustment of tonicity such as sodium chloride or dextrose may also be a carrier. This term expressly incorporates additional carriers provided in the Formulations and Use section provided below.
  • compositions include those salts of a pharmaceutically acceptable composition formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2- ethylamino ethanol, histidine, and procaine. If the composition is basic, salts may be prepared from pharmaceutically acceptable non-toxic acids including inorganic and organic acids.
  • Such acids include acetic, benzene-sulfonic (besylate), benzoic, camphorsulfonic, citric, ethenesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric acid, p-toluenesulfonic, and the like. Particularly preferred are besylate, hydrobromic, hydrochloric, phosphoric and sulfuric acids. If the composition is acidic, salts may be prepared from pharmaceutically acceptable organic and inorganic bases.
  • Suitable organic bases include, but are not limited to, lysine, N,N'- dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine.
  • Suitable inorganic bases include, but are not limited to, alkaline and earth-alkaline metals such as aluminum, calcium, lithium, magnesium, potassium, sodium and zinc. This term expressly incorporates additional salts provided in the Formulations and Use section provided below.
  • Pro-drug As used herein, the term "pro-drug" refers to any composition which releases an active drug in vivo when such a composition is administered to a mammalian subject.
  • Pro-drugs can be prepared, for example, by functional group modification of a parent drug.
  • the functional group may be cleaved in vivo to release the active parent drug compound.
  • Pro-drugs include, for example, compounds in which a group that may be cleaved in vivo is attached to a hydroxy, amino or carboxyl group in the active drug.
  • Examples of pro-drugs include, but are not limited to esters (e.g., acetate, methyl, ethyl, formate, and benzoate derivatives), carbamates, amides and ethers. Methods for synthesizing such pro-drugs are known to those of skill in the art.
  • therapeutically effective amount refers to those amounts that, when administered to a particular subject in view of the nature and severity of that subject's disease or condition, will have a desired therapeutic effect, e.g., an amount which will cure, prevent, inhibit, or at least partially arrest or partially prevent a target disease or condition. More specific embodiments are included in the Pharmaceutical Preparations and Methods of Administration section below.
  • a Novel Nitration Pathway Provides a New Target for Pain and Inflammation Treatment The present invention provides methods for improving pain and/or nitration inhibitors as well as reducing the amount necessary to produce analgesia in subjects in need of pain relief.
  • references to pain and/or nitration inhibitors below includes combinations of multiple pain inhibitors, multiple nitration inhibitors, and multiple pain and nitration inhibitors.
  • one aspect on the invention provides a method for providing a service.
  • the present invention provides a method of increasing revenues from a nitration and/or pain inhibitor.
  • a method for increasing the value of a specific pain inhibitor and/or nitration inhibitor is provided.
  • the present invention provides a method for generating revenue based on pain and/or nitration inhibitors.
  • the fee is received as a royalty payment.
  • the fee is received through sales of seed generated by the new pain reliever combination.
  • the fee is based on sales of a product made from the new pain reliever.
  • the fee is based on licensing of the new pain reliever or product.
  • the fee is based on the receipt of products or services.
  • the present invention provides a method for performing a service.
  • the present invention provides a method for performing a service for a third party.
  • methods for establishing strategic relationships with a third party can comprise the steps of initiating a research and development program with a third party to develop a pain reliever. If desired, the method can include the step of working with the third party to develop the pain reliever. In an embodiment, the method includes the step of licensing to the third party at least a portion of the technology necessary to develop the pain reliever.
  • the novel combinations can be marketed to end-users and consumers such as hospitals, pharmacists, formularies and subjects in need of pain relief.
  • the present invention provides novel compounds which may act individually or in an additive or synergistic fashion to enhance the treatment of pain. Because known pain relievers, which may have negative side effects when administered alone, can be reduces substantially when administered in cojnbination with the present compounds and compositions, significant efficiencies may be obtained. For example, if the amount of very expensive known compositions required to provide pain relief may be reduced by administering a synergistic combination of that known composition and a composition of the present invention, then drug manufacturers, hospitals, formularies and the end-consumer would be willing to purchase such combinations (assuming the compositions of the present invention are cheaper to purchase and produce than the known composition). In addition, the Food and Drug Administration has recently removed well known pain relievers from the market because they cause harm in humans.
  • the compounds and combinations are based on the following: Numerous studies have shown that glutamate-mediated activation of the N-methyl-D-aspartate (NMDA) receptor is fundamental in the development of hyperalgesia. Overt activation of this receptor release superoxide (SO) and nitric oxide that in turn form peroxynitrite (PN). An important mechanism of SO and PN in disease states is nitration of tyrosine residues in various receptors , enzymes, co-factors, cytokines and other compounds in a subject. However, to date, nitration of receptors, enzymes and other compounds has not been implicated in the development and maintenance of pain or inflammation.
  • the present invention provides novel compounds, compositions, and combinations of nitration inhibitors and pain inhibitors.
  • Applicants invention provides SO-driven and PN-driven nitration as a target for the development and maintenance of central sensitization and hyperalgesia provides a novel route of administration for the treatment of pain and inflammation in subjects suffering from acute or chronic pain. Inflammation is also a key component of pain.
  • Superoxide is a potent pro-inflammatory mediator in, e.g., the release of various cytokines, recruitment of neutrophils at sites of inflammation, poly-ADP-ribose-polymerase (PARP) activation, formation of peroxynitrite (PN) and nitration with deactivation of key proteins including endogenous superoxide dismutase, the enzyme that keeps SO at low levels in vivo.
  • PARP poly-ADP-ribose-polymerase
  • PN peroxynitrite
  • MnSOD, GS and NMDA receptor subunits are examples of key targets in this pathway.
  • compositions which inhibit nitration of SO- mediated PN formation and subsequent nitration are key in the development and maintenance of central sensitization, and when combined with other nitration, pain and/or inflammation inhibitors provides new analgesics and analgesic combinations which overcome the problems associated with NSAID or opioid administration while at the same time enhance the effect of the such known inhibitors to extend the potency and efficacy of the nitration, pain or inflammation inhibitor.
  • this invention provides a method for providing a service comprising the acts of identifying a nitration inhibitor effective to treat, prevent, reverse or inhibit pain in a subject; and preparing a therapeutically effective dosage form of said nitration inhibitor or pro-drug thereof.
  • a method for providing a service comprising the acts of identifying a combination of a nitration inhibitor or pro-drug thereof and a pain inhibitor or pro-drug thereof, said nitration inhibitor being effective to treat, prevent, reverse or inhibit pain in combination with said pain inhibitor in a subject, and such that said combination enhances an analgesic effect of the pain inhibitor in the subject; and preparing a therapeutically effective dosage form of the combination.
  • a method for generating revenue comprising the acts of identifying a nitration inhibitor effective to treat, prevent, reverse or inhibit pain in a subject; and preparing a therapeutically effective dosage form of said nitration inhibitor or pro-drug thereof.
  • a method for generating revenue comprising the acts of identifying a combination of a nitration inhibitor or pro-drug thereof and a pain inhibitor or pro-drug thereof, said nitration inhibitor being effective to treat, prevent, reverse or inhibit pain in combination with said pain inhibitor in a subject, and such that said combination enhances an analgesic effect of the pain inhibitor in the subject; and preparing a therapeutically effective dosage form of the combination.
  • a method for providing a service comprising the acts of identifying a nitration inhibitor effective to reverse opioid tolerance in a subject; and preparing a therapeutically effective dosage form of said nitration inhibitor or pro-drug thereof.
  • the relevant methods above may further comprise marketing the nitration inhibitor or pro-drug thereof in commerce.
  • the relevant methods above may also further comprise selling the nitration inhibitor or pro-drug thereof in commerce.
  • the relevant methods may further comprise marketing the nitration inhibitor, or pro-drug thereof, and pain inhibitor, or pro-drug thereof, combination in commerce.
  • the relevant methods above may also further comprise selling the nitration inhibitor, or pro-drug thereof, and pain inhibitor, or pro-drug thereof, combination in commerce.
  • Nitration inhibitors are commercially available and methods for determining whether a compound or composition is an active nitration inhibitor are provided in the Examples below.
  • the nitration inhibitor is selected from the group consisting of a NOS inhibitor, NO generating system inhibitor, NO scavenger, and any combination thereof.
  • the NOS inhibitor is selected from the group consisting of an eNOS inhibitor, iNOS inhibitor, nNOS inhibitor, and any combination thereof.
  • the eNOS inhibitor is selected from the group consisting of NG-monomethyl-L-arginine (LNMMA), NG nitro-L-arginine (L- NA), NG-nitro-L-arginine methyl ester (L-NAME), and any combination thereof;
  • the iNOS inhibitor is selected from the group consisting of L-N5-(1-iminoethyl)-omithine (L-NIL), aminoguanidine, 1400W (N-3-aminomethyl-benzylacetamidine), and any combination thereof;
  • the nNOS inhibitor is selected from the group consisting of 7-nitroindazole (7- Nl), 3-bromo-7-nitroindazole, 6-nitroindazole, Ng-propyl-L-arginine, and any combination thereof.
  • the NO scavenger is selected from the group consisting of hemoglobin, PTIO, NPO, and any combination thereof.
  • the nitration is initiated in a central sensitization pathway of the subject.
  • the pain is initiated in a central sensitization pathway of the subject.
  • the opioid tolerance is initiated in a central sensitization pathway of the subject.
  • the pain inhibitor is an opioid.
  • the opioid is selected from the group consisting of morphine, oxycontin, oxycodone, codeine, fentanyl and any combination thereof.
  • the pain inhibitor is an NSAID.
  • NSAID is selected from the group consisting of ibuprofen (Advil®), naproxen (Aleve® or Naprosyn®), aspirin (Bayer®), celecoxib (Celebrex®), diclofenac (Voltaren®), etodolac (Lodine®), fenoprofen (Nalfon®), indomethacin (Indocin®), ketoprofen (Orudis® or Oruvail®), ketoralac (Toradol®), oxaprozin (Daypro®), nabumetone (Relafen®), sulindac (Clinoril®), tolmetin (Tolectin®), and rofecoxib (Vioxx®).
  • the nitration inhibitor is selected from the group consisting of a SO generating system inhibitor, SO scavenger, and any combination thereof.
  • the SO generating system inhibitor is a xanthine oxidase inhibitor.
  • the complexed metal of the macrocycle provides for catalytic dismutation (or "scavenging") of superoxide.
  • SO scavenger is represented by the following formula:
  • Rs, R's, Rg, and R' g are independently selected from the group consisting of hydrogen, and substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylcycloalkyl, cycloalkenylalkyl, alkylcycloalkyl, alkylcycloalkenyl, alkenylcycloalkyl, alkenylcycloalkenyl, heterocyclic, aryl, aralkyl, hyaluronic acid, polyethyleneglycol, -OR ⁇ 0 , -NR10R11, -COR10, -C0 2 R ⁇ o, -CONR10R11, -O-(-(CH 2 ) a -O) b -R 10 , -SR 10 , -SOR 10 , -SO 2 R ⁇ 0 ,
  • R 1o and Ru are independently selected from the group consisting of hydrogen and alkyl groups, and a and b are integers independently selected from 1 to 6), and substituents attached to the ⁇ -carbon of ⁇ -amino acids; and (b) optionally, one or more of R or R' and Ri or R' ⁇ , R 2 or R' 2 and R 3 or R' 3l R 4 or R' 4 and R 5 or R' 5 , R 6 or R' 6 and R or R' 7 , or R 8 or R' 8 and R 9 or R' g together with the carbon atoms to which they are attached independently form a substituted or unsubstituted nitrogen containing heterocycle having 2 to 20 carbon
  • the SO scavenger is represented by the following formula:
  • a nitrogen of the macrocycle and two adjacent carbon atoms to which the nitrogen is attached independently form a substituted or unsubstituted, saturated or unsaturated, nitrogen-containing heterocycle W having 2 to 20 carbon atoms, which may be an aromatic heterocycle in which case the hydrogen attached to the nitrogen which is both part of the heterocycle and the macrocycle and the R groups attached to the carbon atoms which are both part of the heterocycle and the macrocycle are absent; and (b) optionally, one or more of R, R 1 f R 2 , R' 2 , R 3 , R' 3 , R , R' 4) R 5 , R' 5 , R 6 , R'e, R 7 ,
  • R' 7 , Rs, R's, Rg, and R' g are independently selected from the group consisting of hydrogen, and substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylcycloalkyl, cycloalkenylalkyl, alkylcycloalkyl, alkylcycloalkenyl, alkenylcycloalkyl, alkenylcycloalkenyl, heterocyclic, aryl, aralkyl, hyaluronic acid, polyethyleneglycol, -OR 10 , -NR 10 R. ⁇ , -COR 10 , -C0 2 R ⁇ o, -CONR 10 Rn, -O-(-(CH 2 ) a -O) b -R ⁇ 0 , -SR10, -SOR10, -S0 2 R ⁇
  • R' 7 , R 8 , R's, Rg, and R' 9 may be bound to an atom of heterocycle W to form a strap represented by the formula: - (CH 2 ), - Q - (CH 2 )j - R - (CH 2 ) K - S - (CH 2 ) L - wherein I, J, K and L independently are integers from 0 to 10 and Q, R and S are independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, alkaryl, alkheteroaryl, aza, amide, ammonium, oxa, thia, sulfonyl, sulfinyl, sulfonamide, phosphoryl, phosphinyl, phosphino, phosphonium, keto, ester, alcohol, carbamate, urea, thiocarbonyl, borates, boranes,
  • R and S are independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, alkaryl, alkheteroaryl, aza, amide, ammonium, oxa, thia, sulfonyl, sulfinyl, sulfonamide, phosphoryl, phosphinyl, phosphino, phosphonium, keto, ester, alcohol, carbamate, urea, thiocarbonyl, borates, boranes, boraza, silyl, siloxy, silaza and combinations thereof; and (h) optionally, one or more of R, Ri, R 2 , R' 2 , R 3 , R 4 , R 5 , R' 5 , R
  • U and V are saturated cycloalkyl heterocycles having 3 to 20 carbon atoms. More preferably, U and V are saturated cycloalkyl heterocycles having 4 to 10 carbon atoms. Still more preferably, U and V are trans-cyclohexanyl fused rings.
  • W is preferably a substituted pyridino moiety. More preferably, U and V are trans-cyclohexanyl fused rings and W is a substituted pyridino moiety.
  • transition metal M is preferably selected from the group consisting of Mn, Fe, Ni and V. M40403, which is within the scope of the description above, is described in Fig.
  • the nitration inhibitor is selected from the group consisting of a peroxynitrite generating system inhibitor, peroxynitrite scavenger, and any combination thereof.
  • the peroxynitrite scavenger is represented by a formula selected from the group of formulas consisting of: Structure I
  • R 3 , R 6 , R 9 and R ⁇ 2 are independently selected from the group consisting of H, alkyl, alkenyl, CH 2 , COOH, phenyl, pyridyl, and N-alkylpyridyl, such that phenyl, pyridyl and N-alkylpyridyl are:
  • N-Alkylpyridyl which are attached at a carbon atom; and wherein Phenyl is optionally substituted by a substituent selected from the group consisting of a halogen, alkyl, aryl, benzyl, COOH, CONH 2 , S0 3 H, N0 2 , NH 2 , N(R) 3+ and NHCOR', wherein R is selected from the group consisting of hydrogen, alkyl, aryl and alkaryl, and R' is alkyl; wherein Pyridyl is optionally substituted by a substituent selected from the group consisting of a halogen, alkyl, aryl, benzyl, COOH, CONH 2 , S0 3 H, N0 2 , NH 2 , N(R) 3+ and NHCOR', wherein R and R' are as defined above; and wherein N-Alkylpyridyl is optionally substituted by a substituent selected from the group consisting of a
  • R' is CH or N
  • R-i, R 2 , R3, R 4 , R5, Re, R7, Rs, Rg, R.o, 111 R12, R.3, R. 4 , R-15, and R16 are independently selected from the group consisting of H, SO 3 H, COOH, N0 2 , NH 2 , and N- alkylamino
  • X, Y, Z, M and n are as defined above; Structure III
  • Ri, R 5 , Rg, and R ⁇ 3 are independently selected from the group consisting of a direct bond and CH 2 ;
  • R 2 , R' 2 , R ) R' , Re, R' ⁇ , Rs, R's, R10, R 10, R ⁇ 2 , R' ⁇ 2 , R ⁇ 4 , R' ⁇ 4 , Ri6, and R'i6 are independently selected from the group consisting of H and alkyl;
  • R 3 , R 7 , Ru, and R i5 are independently selected from the group consisting of H and alkyl; and
  • X, Y, Z, M and n are as defined above;
  • Ri, R 5 , R 8 , and R ⁇ 2 are independently selected from the group consisting of a direct bond and CH 2 ;
  • R 2 , R' 2 , R , R' 4 , Re, R' 6 , R 7 , Rg, R'g, Ru, R'n, R ⁇ s, R' ⁇ s, and Ri 4 are independently selected from the group consisting of H and alkyl;
  • R 3 and R ⁇ 0 are independently selected from the group consisting of H and alkyl; and
  • X, Y, Z, M and n are as defined above;
  • Ri, R 4 , R 8 , and R 2 are independently selected from the group consisting of a direct bond and CH 2 ;
  • R 2 , R' 2 , R 3 , R 5 , R's, R 7 , Rg, R'g, Ru, R' ⁇ ⁇ , R13, R'13 and R are independently selected from the group consisting of H and alkyl;
  • R 10 is H or alkyl; and
  • X, Y, Z, M and n are as defined above;
  • Ri, R 4 , R 7 and R 10 are independently selected from the group consisting of a 10 direct bond and CH 2 ;
  • R2, R'2, R3, R5, R's, Re, Rs, R's, Rg, R-t 1, R'11 and R ⁇ 2 are independently selected from the group consisting of H and alkyl; and X, Y, Z, M and n are as defined above;
  • Ri, R , R 8 and Ru are independently selected from the group consisting of a direct bond and CH 2 ;
  • R2, R3, R'3, Rs, R's, R7, RV, Rg, R10, R'10, R12, R'12 and R13 are independently selected from the group consisting of H and alkyl;
  • 0 R 6 is hydrogen or alkyl;
  • X, Y, Z, M and n are as defined above;
  • 0 are independently selected from the group consisting of H and alkyl;
  • R2, R 3 , R'3, Rs, R's, Re, Rs, Rg, R'g, Rn, R'n and R ⁇ 2 are independently selected from the group consisting of H and alkyl;
  • X, Y, Z, M and n are as defined above;
  • Ri, R 3 , R 4 , and R 6 are independently selected from the group consisting of H and alkyl;
  • R 2 and R 5 are independently selected from the group consisting of H, alkyl, S0 3 H, N0 2 , NH 2 , halogen, COOH and N(R) 3+ wherein R is as defined above; and
  • X, Y, Z, M and n are as defined above;
  • R , R 2 , R 3 , and R 4 are independently selected from the group consisting of H, alkyl, S0 3 H, N0 2 , NH 2 , halogen, COOH and N(R) 3+ wherein R is as defined above; and X, Y, Z, M and n are as defined above; and Structure IV
  • Ri, R , R 2 , R' 2 , R 3 , R' 3 , R*, R' 4 , Rs, R's, R 6 , R'e, R7 and R' 7 are independently selected from the group consisting of H, alkyl, alkoxy, N0 2 , aryl, halogen, NH 2 and S0 3 H, further wherein R 6 , R'e, 7 and R' 7 together with one other of R 6 , R' ⁇ , R 7 and R' 7 optionally form a heterocycle having 5 to 8 carbon atoms and form a ring with the carbon atoms of the macrocycle to which they are attached; M 1 is selected from the group consisting of Fe, Ni or V; and X, Y, Z and n are as defined above.
  • a method wherein the nitration inhibitor is selected from the group consisting of a SO generating system inhibitor, SO scavenger, NOS inhibitor, NO generating system inhibitor, NO scavenger, peroxynitrite generating system inhibitor, peroxynitrite scavenger, and any combination thereof.
  • the pain inhibitor is an opioid or NSAID and the nitration inhibitor is selected from the group consisting of a SO generating system inhibitor, SO scavenger, NOS inhibitor, NO generating system inhibitor, NO scavenger, peroxynitrite generating system inhibitor, peroxynitrite scavenger, and any combination thereof.
  • the subject may be a mammal.
  • the mammal is a human.
  • NMDA N-methyl-D- aspartate
  • NMDA-receptor activation releases superoxide the formation of which has been closely associated with numerous glutamate-mediated actions including neurotoxicity and stroke.
  • MnSOD manganese superoxide dismutase
  • SOD2 endogenous manganese superoxide dismutase
  • Nitration of the endogenous SOD enzyme is carried out by peroxynitrite, the reaction product of superoxide and nitric oxide.
  • NMDA-receptor activation favors the accumulation of peroxynitrite by forming superoxide and nitric oxide simultaneously.
  • nitration destroys the enzymatic activity of MnSOD, an event favoring the accumulation of superoxide (the enzyme can no longer dismute and hence remove superoxide) and superoxide-driven damage.
  • the associations of many glutamate actions with the formation of superoxide suggest that superoxide is an important mediator of NMDA-mediated hyperalgesia and subsequently that nitration of the endogenous MnSOD enzyme is an important pathway by which superoxide modulates the hyperalgesic response to NMDA.
  • M40403 (Fig. 1 A), a synthetic and selective superoxide dismutase mimetic.
  • M40403 is a stable, low molecular weight, manganese-containing, non-peptidic molecule possessing the function and catalytic rate of native superoxide dismutase enzymes, but with the advantage of being a much smaller molecule (MW 483 vs MW 30,000 for the mimetic and native enzyme, respectively) was used to elucidate this novel pathway. Furthermore, M40403 is stable in vivo, penetrates cells readily, has wide tissue distribution in rats, is excreted intact with no detectable dissociation and is recovered in urine and feces intact. M40404 (Fig. 1 B) the inactive congener of M40403 has no SOD catalytic activity and is pharmacologically inactive in vivo in models of inflammation.
  • the present invention provides that superoxide formation is a critical step in NMDA-mediated hyperalgesia and that superoxide-driven nitration of endogenous MnSOD is a critical pathway in the response.
  • Such superoxide-driven nitration when targeted by the compounds of the present invention may be used to treat, prevent, reverse and inhibit such hyperalgesia.
  • Important pro-inflammatory roles for superoxide include: endothelial cell damage and increased microvascular permeability, release of cytokines, recruitment of neutrophils at sites of inflammation, single-strand DNA damage and poly-ADP-ribose- polymerase (PARP) activation.
  • PARP poly-ADP-ribose- polymerase
  • the present invention provides that superoxide rapidly combines with nitric oxide removing an important homeostatic signaling molecule and at the same time forming peroxynitrite (PN), a potent cytotoxic and pro-inflammatory agent.
  • PN peroxynitrite
  • the invention provides that removal of superoxide by synthetic superoxide dismutase mimetics such as Mn(lll)tetrakis (4-benzoic acid) porphyrin (MnTBAP), M40403 and peroxynitrite by decomposition catalysts such as 5,10,15,20-tetrakis(2,4,6-trimethyl-3,5-disulfonatophenyl)porphyrinato iron III (FeTMPS) are shown to be affective in treating, preventing, reversing or inhibiting pain, and in particular acute inflammatory pain.
  • synthetic superoxide dismutase mimetics such as Mn(lll)tetrakis (4-benzoic acid) porphyrin (MnTBAP), M40403
  • peroxynitrite such as 5,10,15,20-tetrakis(2,4,6-trimethyl-3,5-disulfonatophenyl)porphyrinato iron III (FeTMPS) are shown
  • compositions treat, prevent, reverse, inhibit, control, or at least partially arrest or partially prevent, pain associated with superoxide-induced nitration of enzymes, receptors or other compounds in a subject and can be administered to a subject at therapeutically effective doses for the inhibition, prevention, prophylaxis or therapy for such pain caused by superoxide-induced nitration.
  • the compositions of the present invention comprise a therapeutically effective dosage of a nitration inhibitor which includes, e.g., nitration inhibitors, alone or in combination with another or a known nitration, pain or inflammation inhibitors.
  • compositions of the present invention which includes therapeutically, inhibitory, preventive and prophylactically effective doses of the compositions of the present invention and is more particularly defined below.
  • the subject is preferably an animal, including, but not limited to, mammals, reptiles and avians, more preferably horses, cows, dogs, cats, sheep, pigs, and chickens, and most preferably human.
  • compositions that exhibit large therapeutic indices are preferred. While compositions exhibiting toxic side effects may be used, care should be taken to design a delivery system that targets such compositions to the site affected by the disease or disorder in order to minimize potential damage to unaffected cells and reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosages for use in humans and other mammals.
  • the dosage of such compositions lies preferably within a range of circulating plasma or other bodily fluid concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dosage may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (the concentration of the test composition that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful dosages in humans and other mammals.
  • Composition levels in plasma may be measured, for example, by high performance liquid chromatography.
  • compositions that may be combined with pharmaceutically acceptable carriers to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. It will be appreciated by those skilled in the art that the unit content of a composition contained in an individual dose of each dosage form need not in itself constitute a therapeutically effective amount, as the necessary therapeutically effective amount could be reached by administration of a number of individual doses. The selection of dosage depends upon the dosage form utilized, the condition being treated, and the particular purpose to be achieved according to the determination of those skilled in the art.
  • the dosage regime for treating a disease or condition with the compositions and/or composition combinations of this invention is selected in accordance with a variety of factors, including the type, age, weight, sex, diet and medical condition of the patient, the route of administration, pharmacological considerations such as activity, efficacy, pharmacokinetic and toxicology profiles of the particular composition employed, whether a composition delivery system is utilized and whether the composition is administered as a pro-drug or part of a drug combination.
  • the dosage regime actually employed may vary widely from subject to subject.
  • compositions of the present invention may be formulated by known methods for administration to a subject using several routes which include, but are not limited to, parenteral, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and ophthalmic routes.
  • the individual compositions may also be administered in combination with one or more additional compositions of the present invention and/or together with other biologically active or biologically inert agents ("composition combinations").
  • Such biologically active or inert agents may be in fluid or mechanical communication with the composition(s) or attached to the composition(s) by ionic, covalent, Van der Waals, hydrophobic, hydrophilic or other physical forces.
  • compositions or composition combinations may be formulated by any conventional manner using one or more pharmaceutically acceptable carriers and/or excipients.
  • the compositions and their pharmaceutically acceptable salts and solvates may be specifically formulated for administration, e.g., by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral or rectal administration.
  • the composition or composition combinations may take the form of charged, neutral and/or other pharmaceutically acceptable salt forms.
  • pharmaceutically acceptable carriers include, but are not limited to, those described in REMINGTON'S PHARMACEUTICAL SCIENCES (A.R.
  • compositions may also take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, controlled- or sustained-release formulations and the like.
  • Such compositions will contain a therapeutically effective amount of the composition, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
  • the formulation should suit the mode of administration.
  • composition or composition combination may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form in ampoules or in multi-dose containers with an optional preservative added.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass, plastic or the like.
  • the composition may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • a parenteral preparation may be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent (e.g., as a solution in 1,3- butanediol).
  • a nontoxic parenterally acceptable diluent or solvent e.g., as a solution in 1,3- butanediol.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid may be used in the parenteral preparation.
  • the composition may be in powder form for constitution with a suitable vehicle, such as sterile pyrogen-free water, before use.
  • a composition suitable for parenteral administration may comprise a sterile isotonic saline solution containing between 0.1 percent and 90 percent weight per volume of the composition or composition combination.
  • a solution may contain from about 5 percent to about 20 percent, more preferably from about 5 percent to about 17 percent, more preferably from about 8 to about 14 percent, and still more preferably about 10 percent of the composition.
  • the solution or powder preparation may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
  • Other methods of parenteral delivery of compositions will be known to the skilled artisan and are within the scope of the invention.
  • composition or composition combination may take the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents, fillers, lubricants and disintegrants:
  • Binding agents include, but are not limited to, corn starch, potato starch, or other starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pre- gelatinized starch, hydroxypropyl methyl cellulose, (e.g., Nos. 2208, 2906, 2910), microcrystalline cellulose, and mixtures thereof.
  • natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl
  • Suitable forms of microcrystalline cellulose include, for example, the materials sold as AVICEL-PH-101 , AVICEL-PH- 103 and AVICEL-PH-105 (available from FMC Corporation, American Viscose Division, Avicel Sales, Marcus Hook, Pennsylvania, USA).
  • An exemplary suitable binder is a mixture of microcrystalline cellulose and sodium carboxymethyl cellulose sold as AVICEL RC-581 by FMC Corporation.
  • Fillers include, but are not limited to, talc, calcium carbonate (e.g., granules or powder), lactose, microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof.
  • Lubricants include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laurate, agar, and mixtures thereof.
  • Additional lubricants include, for example, a syloid silica gel (AEROSIL 200, manufactured by W.R. Grace Co.
  • Disintegrants include, but are not limited to, agar-agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, other starches, pre-gelatinized starch, other starches, clays, other algins, other celluloses, gums, and mixtures thereof.
  • the tablets or capsules may optionally be coated by methods well known in the art. If binders and/or fillers are used with the compositions of the invention, they are typically formulated as about 50 to about 99 weight percent of the composition. Preferably, about 0.5 to about 15 weight percent of disintegrant, preferably about 1 to about 5 weight percent of disintegrant, may be used in the composition. A lubricant may optionally be added, typically in an amount of less than about 1 weight percent of the composition. Techniques and pharmaceutically acceptable additives for making solid oral dosage forms are described in Marshall, SOLID ORAL DOSAGE FORMS, Modern Pharmaceutics (Banker and Rhodes, Eds.), 7:359-427 (1979). Other less typical formulations are known in the art.
  • Liquid preparations for oral administration may take the form of solutions, syrups or suspensions. Alternatively, the liquid preparations may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and/or preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
  • suspending agents e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats
  • emulsifying agents e.g., lecithin or acacia
  • non-aqueous vehicles e.g., almond oil, oily esters, eth
  • compositions of the present invention may also contain buffer salts, flavoring, coloring, perfuming and sweetening agents as appropriate.
  • Preparations for oral administration may also be formulated to achieve controlled release of the composition.
  • Oral formulations preferably contain 10% to 95% composition.
  • the compositions of the present invention may be formulated for buccal administration in the form of tablets or lozenges formulated in a conventional manner. Other methods of oral delivery of compositions will be known to the skilled artisan and are within the scope of the invention.
  • Controlled-Release Administration may be formulated to extend the activity of the composition or composition combination and reduce dosage frequency. Controlled-release preparations can also be used to effect the time of onset of action or other characteristics, such as blood levels of the composition, and consequently affect the occurrence of side effects. Controlled-release preparations may be designed to initially release an amount of a composition that produces the desired therapeutic effect, and gradually and continually release other amounts of the composition to maintain the level of therapeutic effect over an extended period of time. In order to maintain a near-constant level of a composition in the body, the composition could be released from the dosage form at a rate that will replace the amount of composition being metabolized and/or excreted from the body.
  • Controlled-release of a composition may be stimulated by various inducers, e.g., change in pH, change in temperature, enzymes, water, or other physiological conditions or molecules.
  • Controlled-release systems may include, for example, an infusion pump which may be used to administer the composition in a manner similar to that used for delivering insulin or chemotherapy to specific organs or tumors.
  • the composition is administered in combination with a biodegradable, biocompatible polymeric implant that releases the composition over a controlled period of time at a selected site.
  • polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, and copolymers and blends thereof.
  • compositions of the invention may be administered by other controlled-release means or delivery devices that are well known to those of ordinary skill in the art. These include, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or the like, or a combination of any of the above to provide the desired release profile in varying proportions. Other methods of controlled-release delivery of compositions will be known to the skilled artisan and are within the scope of the invention.
  • compositions or composition combination may also be administered directly to the lung by inhalation.
  • a composition may be conveniently delivered to the lung by a number of different devices.
  • a Metered Dose Inhaler which utilizes canisters that contain a suitable low boiling point propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas may be used to deliver a composition directly to the lung.
  • MDI devices are available from a number of suppliers such as 3M Corporation, Aventis, Boehringer Ingleheim, Forest Laboratories, Glaxo-Wellcome, Schering Plough and Vectura.
  • a Dry Powder Inhaler (DPI) device may be used to administer a composition to the lung.
  • DPI devices typically use a mechanism such as a burst of gas to create a cloud of dry powder inside a container, which may then be inhaled by the patient.
  • DPI devices are also well known in the art and may be purchased from a number of vendors which include, for example, Fisons, Glaxo-Wellcome, Inhale Therapeutic Systems, ML Laboratories, Qdose and Vectura.
  • MDDPI multiple dose DPI
  • MDDPI devices are available from companies such as AstraZeneca, GlaxoWellcome, IVAX, Schering Plough, SkyePharma and Vectura.
  • capsules and cartridges of gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch for these systems.
  • a liquid spray device supplied, for example, by Aradigm Corporation.
  • Liquid spray systems use extremely small nozzle holes to aerosolize liquid composition formulations that may then be directly inhaled into the lung.
  • a nebulizer device may be used to deliver a composition to the lung.
  • Nebulizers create aerosols from liquid composition formulations by using, for example, ultrasonic energy to form fine particles that may be readily inhaled.
  • nebulizers include devices supplied by Sheffield/Systemic Pulmonary Delivery Ltd., Aventis and Batelle Pulmonary Therapeutics.
  • an electrohydrodynamic (“EHD") aerosol device may be used to deliver a composition to the lung.
  • EHD aerosol devices use electrical energy to aerosolize liquid composition solutions or suspensions.
  • the electrochemical properties of the composition formulation are important parameters to optimize when delivering this composition to the lung with an EHD aerosol device. Such optimization is routinely performed by one of skill in the art.
  • Other methods of intra-pulmonary delivery of compositions will be known to the skilled artisan and are within the scope of the invention.
  • Liquid composition formulations suitable for use with nebulizers and liquid spray devices and EHD aerosol devices will typically include the composition with a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier is a liquid such as alcohol, water, polyethylene glycol or a perfluorocarbon.
  • another material may be added to alter the aerosol properties of the solution or suspension of the composition.
  • this material may be a liquid such as an alcohol, glycol, polyglycol or a fatty acid.
  • Other methods of formulating liquid composition solutions or suspensions suitable for use in aerosol devices are known to those of skill in the art.
  • compositions or composition combination may also be formulated as a depot preparation.
  • Such long-acting formulations may be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection.
  • the compositions may be formulated with suitable polymeric or hydrophobic materials such as an emulsion in an acceptable oil or ion exchange resins, or as sparingly soluble derivatives such as a sparingly soluble salt.
  • suitable polymeric or hydrophobic materials such as an emulsion in an acceptable oil or ion exchange resins, or as sparingly soluble derivatives such as a sparingly soluble salt.
  • Other methods of depot delivery of compositions will be known to the skilled artisan and are within the scope of the invention.
  • composition or composition combination may be combined with a carrier so that an effective dosage is delivered, based on the desired activity ranging from an effective dosage, for example, of 1.0 ⁇ M to 1.0 mM.
  • a topical composition is applied to the skin.
  • the carrier may be in the form of, for example, and not by way of limitation, an ointment, cream, gel, paste, foam, aerosol, suppository, pad or gelled stick.
  • a topical formulation may also consist of a therapeutically effective amount of the composition in an ophthalmologically acceptable excipient such as buffered saline, mineral oil, vegetable oils such as corn or arachis oil, petroleum jelly, Miglyol 182, alcohol solutions, or liposomes or liposome-like products.
  • an ophthalmologically acceptable excipient such as buffered saline, mineral oil, vegetable oils such as corn or arachis oil, petroleum jelly, Miglyol 182, alcohol solutions, or liposomes or liposome-like products.
  • ophthalmologically acceptable excipient such as buffered saline, mineral oil, vegetable oils such as corn or arachis oil, petroleum jelly, Miglyol 182, alcohol solutions, or liposomes or liposome-like products.
  • Any of these compositions may also include preservatives, antioxidants, antibiotics, immunosuppressants, and other biologically or pharmaceutically effective agents which do not exert a detrimental effect on the composition.
  • composition or composition combination may also be formulated in rectal formulations such as suppositories or retention enemas containing conventional suppository bases such as cocoa butter or other glycerides and binders and carriers such as triglycerides, microcrystalline cellulose, gum tragacanth or gelatin.
  • Suppositories may contain the composition in the range of 0.5% to 10% by weight.
  • Other methods of suppository delivery of compositions will be known to the skilled artisan and are within the scope of the invention.
  • compositions of the invention are known in the art and can be used to administer the compositions of the invention. Moreover, these and other delivery systems may be combined and/or modified to optimize the administration of the compositions of the present invention. Exemplary formulations using the compositions of the present invention are described below in Example 4 (the compositions of the present invention are indicated as the active ingredient, but those of skill in the art will recognize that pro-drugs and composition combinations are also meant to be encompassed by this term).
  • compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition.
  • the pack may for example comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • Example 1 Treatments for Pain Male Sprague-Dawley rats (250-260 g, Raleigh, NC, USA) were purchased with intrathecally-implanted cannulas (32 gauge, polyurethane) and used for these studies. All animals were housed and cared for in accordance with the guidelines of the Institutional Animal Care and Use Committee and in accordance with NIH guidelines on laboratory animal welfare.
  • the active SOD mimetic M40403 and its inactive congener, M40404 were synthesized as previously described (Salvemini D, Wang Z-Q, Zweier JL, Samouilov A, Macarthur H, Misko TP, Currie MG, Cuzzocrea S, Sikorski JA and Riley DP (1999). "A non- peptidyl mimic of superoxide dismutase with therapeutic activity in rats.” Science 286: 304- 306.). Unless specified, all materials were purchased from Sigma-Aldrich Corp. (St Louis, MO).
  • the dose used was selected from previous studies where it was shown that at this dose and in vivo superoxide-mediated damage in rats was completely blocked by M40403 and not by M40404 (Coppey LJ, Gellett JS, Davidson EP, Dunlap JA, Lund DD, Salvemini D and Yorek MA (2001).
  • Lumbar spinal cord enlargements (L4/L6) were homogenized with lysis buffer with a
  • the lysis buffer (20 mM Tris-base, 150 mM NaCI, 10 % glycerol, 0.1% Triton- X-100, 1% Chaps, 2 mM EGTA) contained 1% protease inhibitor cocktail (v/v). Solubilized extracts were sonicated (5 min.) using a Sonicator (Fisher Scientific) and after 10 min of incubation in ice the lysates were centrifuged (12500 g, 30 min at 4 °C). These supernatants were stored immediately at -80 °C. Protein concentration was determined using the Bicinchoninic Acid (BCA) protein assay (Pierce).
  • BCA Bicinchoninic Acid
  • the mixture of the beads-antibody and binding proteins were resuspended in 50 ⁇ of sample buffer [2x, 0.5 M Tris-HCI, (pH 6.8) 2.5% glycerol/0.5%SDS/200 mM 2-mercaptoethanol/0.001% bromophenol blue], heated to 95 °C (5 min).
  • sample buffer [2x, 0.5 M Tris-HCI, (pH 6.8) 2.5% glycerol/0.5%SDS/200 mM 2-mercaptoethanol/0.001% bromophenol blue]
  • the samples were then loaded in 12% SDS-PAGE mini-gels (Bio-Rad). Rat brain treated with peroxynitrite (Upstate Biotechnology) was used as positive control.
  • Tissue samples were homogenized with 10 mM phosphate buffered saline (pH 7.4) in a Polytron homogenizer and then sonicated on ice for 1 min. (20 s, 3 times). The sonicated samples were subsequently centrifuged at 1 ,100 g for 10 min. SOD activity was measured in the supematants as described (Beauchamp C and Fridovich I (1971).
  • a competitive inhibition assay was performed which used xanthine-xanthine oxidase- generated superoxide to reduce nitroblue tetrazolium (NBT) to blue tetrazolium salt.
  • the reaction was performed in sodium carbonate buffer (50 mM, pH 10.1) containing EDTA (0.1 mM), nitroblue tetrazolium (25 ⁇ M; Sigma-Aldrich Corp., Milan, Italy), xanthine and xanthine- oxidase (0.1 mM and 2 nM respectively; Boehringer, Germany).
  • the rate of NBT reduction was monitored spectrophotometrically (Perkin Elmer Lambda 5 Spectrophotometer, Milan, Italy) at 560 nm.
  • the amount of protein required to inhibit the rate of NTB reduction by 50% was defined as one unit of enzyme activity.
  • Cu,Zn-SOD activity was inhibited by performing the assay in the presence of 2 mM NaCN after pre-incubation for 30 min. Enzymatic activity was expressed in units per milligram of protein
  • an important pathway through which superoxide modulates disease is by forming peroxynitrite, which in turn nitrates and deactivates endogenous MnSOD, an event critical in maintaining and in sustaining high levels of superoxide.
  • this pathway operates in central sensitization (as stated earlier NMDA-receptor activation releases both nitric oxide and superoxide, providing the source of peroxynitrite).
  • MnSOD was found to be nitrated in the spinal cord as shown by immunoprecipitation (Fig. 5, panel A). Furthermore, once nitrated, endogenous MnSOD is deactivated losing its ability to remove superoxide (Fig. 5, panel C).
  • excitatory amino acids including glutamate
  • stimulation of the nociceptors induces the firing of the C-fibers (unmyelinated slowly- conducting afferent) leading to the release of excitatory amino acids, including glutamate, that in turn act on the NMDA-receptor in the spinal cord.
  • C-fibers unmyelinated slowly- conducting afferent
  • NMDA receptor activation is believed to induce the spinal cord neurons to become more sensitive to all of their inputs, resulting in central sensitization seen in acute inflammatory pain and neuropathic pain. Similar mechanisms may be an important part of the development of opioid tolerance that is also blocked by NMDA-receptor antagonists.
  • the present invention provides, for the first time, that superoxide is an important mediator of spinal (i.e., central nervous system or "CNS")hyperalgesia.
  • CNS central nervous system
  • synthetic SOD mimetics and other compounds and compositions are useful agents for the management of pain of various etiologies.
  • the present invention reveals a mechanism by which superoxide modulates NMDA- mediated hyperalgesia (Figs. 6 and 12).
  • MnSOD hyperoxide dismutase mimetic
  • the present invention also provides that nitration of this enzyme is also important in central sensitization in addition to being associated closely to those disease states driven by overt production of superoxide (as the enzyme is now no longer able to dismute superoxide), for example, ischemia and reperfusion, organ transplantation, shock and inflammation. .
  • the present invention provides at least two pathways which can lead to protein nitration. One uses peroxynitrite, (the reaction product of superoxide and nitric oxide) and the other uses hydrogen peroxide and myeloperoxidase. Without being bound to a particular theory, the involvement of these pathways in a particular setting can be dissected pharmacologically by the use of agents which remove superoxide or nitric oxide.
  • nitric oxide inhibits as shown by numerous investigators the formation of peroxynitrite and peroxynitrite- mediated protein nitration.
  • MnSOD nitration was blocked by M40403 supports the role of peroxynitrite and not hydrogen peroxide/myeloperoxidase in this process.
  • nitric oxide has been implicated as a central mediator of NMDA-driven central sensitization.
  • NMDA-mediated hyperalgesia is blocked by nitric oxide synthase inhibitors. Based on the data provided in the present invention, it is believed that inhibition of NMDA-mediated hyperalgesia by various nitric oxide synthase inhibitors is in fact due to inhibition of peroxynitrite-mediated MnSOD nitration. Thus, formation of peroxynitrite and subsequent nitration of spinal MnSOD is a novel pathway of NMDA-mediated hyperalgesia and a new target which can be affected by the compounds and compositions of the present invention. The broader implication of our findings is that superoxide may contribute to various forms of centrally induced hyperalgesia that are driven by NMDA-receptor activation. Thus, superoxide is a therapeutic target for the development of novel analgesics and analgesic combinations (e.g., additive and synergistic combinations).
  • analgesics and analgesic combinations e.g., additive and synergistic combinations.
  • Example 2 Treatments of Pain Due to Inflammation
  • Tail flick and hot plate tests Nociceptive testing was performed by placing the distal third of the tail of a rat in a water bath maintained at 52 °C (tail flick) or by placing rats on a metal surface maintained at 52 °C.
  • the latency to withdrawal (tail flick) or to jumping/licking of a hindpaw (hot plate) was measured once before (control) and at selected time intervals after drug injection.
  • a cut-off latency of 10 seconds is employed to prevent tissue injury.
  • Groups of at least 6 rats each received a subcutaneous injection of SODm (10 mg/kg) or an equivalent volume of vehicle and testing was performed 15, 30, 45 and 60 minutes after injection of the drug or vehicle. Determination of anti-nociception was assessed between 7:00 and 10:00 AM.
  • Decomposed peroxynitrite was obtained by exposing the stock solution of peroxynitrite to air for at least 30 minutes. Drugs or vehicle were given intravenously (2ml/kg) 15 minutes before injection of superoxide and peroxynitrite. Hyperalgesic responses to heat were measured at time of maximal hyperalgesic effect. Time course studies revealed that maximal hyperalgesia was reached at 20 and 30 minutes respectively, for superoxide and peroxynitrite.
  • Drugs or vehicle were administered intravenously (2 ml/kg), subcutaneously (1 ml/kg) or intrathecally (in 10 ⁇ l) at 2 hours post-carrageenan injection (therapeutic treatment) and testing performed 0.25, 0.5, 0.75, 1 , 2 and 3 hours after drug injection. In some experiments, drugs or vehicle were also given 30 minutes before carrageenan (prophylactic treatment) and testing performed every hour up to 5 hours post carrageenan. Changes in paw-volume were measured as previously described. Briefly, paw-volume was measured with a plethysmometer (Ugo-Basile, Varese, Italy) immediately prior to the injection of carrageenan and thereafter at hourly intervals for 5 hours. Edema was expressed as the increase in paw-volume (ml) after carrageenan injection relative to the pre-injection value for each animal. Results are expressed as Paw-volume change (ml).
  • Cytokines (TNF- ⁇ , IL-1 ?, IL-6) released in the paw exudates were measured by ELISA as described previously. Briefly, at 5 hours following the intraplantar injection of carrageenan, rats were sacrificed, and each paw was cut at the level of the calcaneus bone. Paws were gently centrifuged at 250 x g for 20 min in order to recover a sample of the edematous fluid. The volume of fluid recovered from each paw was measured. Cytokines were measured by ELISA (R&D systems, Minneapolis, MN) and results expressed in pg/paw, normalizing values to the amount of exudates recovered from each paw. All determinations were performed in duplicate. Previous studies indicated that maximal levels of these cytokines were seen at 5 hours post carrageenan. Therefore, animals were sacrificed at these time points.
  • Sections were incubated overnight with 1) anti-nitrotyrosine rabbit polyclonal antibody (1 :500 in PBS) and with anti- poly (ADP-Ribose) goat policlonal antibody rat (1:500 in PBS). Sections were washed with PBS, and incubated with secondary antibody (FITC-conjugated anti-rabbit and with TRIC- conjugated anti-goat; Jackson, West Grove, PA) for 2 h at RT. Sections were washed as before, mounted with 90% glycerol in PBS, and observed with a Nikon RCM8000 confocal microscope equipped with a 40X oil objective.
  • Sections were blocked for 1 hr with 100% normal horse serum (Vector), followed by application of monoclonal anti-nitrotyrosine antibody (1 :100 in 100% normal horse serum) at 4 °C overnight in a moist chamber. Treatment with secondary antibody, A/B complex, and DAB were performed by the manufacturer's instructions (Vector ABC Elite Kit, Vector Laboratories).
  • Protein concentration was determined using the Bicinchoninic Acid (BCA) protein assay (Pierce). 4 mg of the solubilized proteins in 500 ⁇ l lysate buffer were incubated with 10 ⁇ g of agarose-conjugated anti-nitrotyrosine antibody (Upstate Biotechnology) for 2 h at room temperature (RT). Agarose beads were collected by centrifugation (1 min at 12000 x g at 4 °C) and washed in PBS (pH 7.4) three times.
  • BCA Bicinchoninic Acid
  • blots were incubated with rabbit polyclonal anti-Mn-SOD (2 hr, RT, 1:1 ,000 dilution; Upstate Biotechnology). After washing with TBS/T, the membranes were incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:10,000 dilution; Amersham) and the specific complex was detected by an enhanced chemiluminescence detection system (ECL, Amersham).
  • ECL enhanced chemiluminescence detection system
  • a competitive inhibition assay was performed which used xanthine-xanthine oxidase-generated SO to reduce nitroblue tetrazolium (NBT) to blue tetrazolium salt.
  • the reaction was performed in sodium carbonate buffer (50 mM, pH 10.1) containing EDTA (0.1 mM), nitroblue tetrazolium (25 ⁇ M; Sigma, Milan, Italy), xanthine and xanthine-oxidase (0.1 mM and 2 nM respectively; Boehringer, Germany).
  • the rate of NBT reduction was monitored spectrophotometrically (Perkin Elmer Lambda 5 Spectrophotometer, Milan, Italy) at 560 nm.
  • the amount of protein required to inhibit the rate of NTB reduction by 50% was defined as one unit of enzyme activity.
  • Cu.Zn- SOD activity was inhibited by performing the assay in the presence of 2 mM NaCN after pre- incubation for 30 min. Enzymatic activity was expressed in units per milligram of protein.
  • Rotarod Test Rats were placed on a rotating rod (7 cm diameter) turning at 10 rpm. The animals were exposed to the rotarod for one session of 180 seconds each day for 3 days to adapt the rats to the apparatus. The rats were then injected subcutaneously with SODm (100 mg/kg) and tested again on the rotarod for periods of approximately 180 sec at selected time intervals.
  • Example 2 Results Utilizing a well-established model of acute inflammatory hyperalgesia, (carrageenan- induced inflammation and hyperalgesia) the role of superoxide in nociception associated with acute inflammation was investigated.
  • Hyperalgesic responses defined as augmented pain intensity in response to painful stimuli seen upon intraplantar injection of carrageenan involve central and peripheral sensitization.
  • Intraplantar injection of carrageenan leads to a time dependent development of inflammation and hyperalgesia, which peaks within 2-3 hours and lasts for a subsequent 6-8 hours.
  • Administration of M40403 (1-10 mg/kg, n 6) prior to carrageenan administration inhibited in a dose-dependent manner the development of edema and thermal hyperalgesia at all time points.
  • Dose-dependent inhibition curves for inflammation and pain taken at 5 h after carrageenan are shown in Fig. 7a.
  • M40403 prevented carrageenan-induced edema and hyperalgesia, in part by inhibiting superoxide- driven cytokine production.
  • the potential participation of peroxynitrite in superoxide-mediated nociception was evaluated initially by immunohistochemical detection of nitrated proteins and PARP activation and subsequently by evaluating whether peroxynitrite promotes hyperalgesia and whether it's removal inhibits acute inflammatory hyperalgesia.
  • nitrated proteins are readily detected in the inflamed paw and are associated with PARP activation (Fig. 8a).
  • the formation of nitrated proteins and PARP activation were blocked by M40403 (Fig. 8a).
  • M40403 when given forty minutes prior to formalin at doses failed to inhibit this phase failed to inhibit this phase (not shown).
  • paw licking time (sec) evoked by formalin in control mice was reduced from 63 ⁇ 5 to 45 ⁇ 2, 15 ⁇ 3, 6 ⁇ 2 and 2 ⁇ 0 in the presence of 0.3, 1 , 3 and 10 mg/kg M40403 respectively.
  • Example 2 Discussion The present invention provides that superoxide is a newly identified mediator of pain, providing an opportunity for novel pain management.
  • the picture depicted in Fig. 12 summarizes the key findings of this investigation. It is well appreciated that during tissue injury and inflammation, hyperalgesia results from a persistent state of peripheral afferent sensitization that subsequently initiates spinal sensitization through the release of the excitatory amino acid, glutamate.
  • the mechanism(s) are complex in nature and involve peripherally and spinally formed inflammatory mediators including peptides, prostanoids, nitric oxide, cytokines, excitatory amino acids (glutamate) as well as spinal cord glial cells activation.
  • the present invention provides that superoxide is formed and plays a major role in the development of pain through direct peripheral sensitization, by promoting inflammation, and by favouring nitration of endogenous MnSOD in the spinal cord.
  • anti-nociceptive effect of M40403, and like compounds includes overall inhibition of the effects of superoxide exerted peripherally and centrally.
  • M40403 exerted a profound anti-inflammatory effect which included inhibition of edema, cytokine release, formation of peroxynitrite and PARP activation. Furthermore, it was noted that by inhibiting inflammation, M40403 inhibited hyperalgesia.
  • a mechanism by which M40403 blocks cytokine release is by preventing the activation of REDOX sensitive transcription factors including NF- ⁇ B and AP-1 by superoxide which in turn regulates the genes that encode (amongst numerous things) various pro-inflammatory and pronociceptive cytokines.
  • REDOX sensitive transcription factors including NF- ⁇ B and AP-1 by superoxide which in turn regulates the genes that encode (amongst numerous things) various pro-inflammatory and pronociceptive cytokines.
  • the same inflammatory molecules play a role in pain as shown by their ability to directly induce hyperalgesia.
  • these cytokines participate in the induction of pain by peripheral nerve inflammation and trauma, subcutaneous inflammation and spinal dynorphin.
  • NAD + functions as a cofactor in glycolysis and the tricarboxylic acid cycle
  • NAD + depletion leads to a rapid fall in intracellular ATP and, ultimately, cell injury.
  • PARP activation has been implicated for instance in the development of hyperalgesia seen upon chronic use of opioids. Furthermore, substantial evidence provided by the utility of PARP inhibitors exists to support that PARP activation is important in inflammation. SOD mimetics of the M40403 class reduce PARP activation in models of acute and chronic inflammation supporting the role of superoxide in this pathway.
  • NMDA N-methyl- D-aspartate
  • MnSOD is a newly identified mediator of pain (Fig. 12) and that its removal by low molecular weight synthetic enzymes of superoxide dismutase represents a therapeutic target for the development of novel non- narcotic analgesics including those compounds and compositions of the present invention.
  • Example 3 - The compositions can be provided in combination with sufficient amounts of known nitration, pain or inflammation inhibitors to elicit a synergistic response in a subject. Synergy may be measured by comparing the baseline pain inhibition by the rat model above and comparing composition combinations. For example, a protocol to determine synergistic combinations includes: . _ -, , -» w • • --.---. J
  • a radiant heat source was focused onto the plantar surface of the affected paw of nerve-injured or carageenan-injected rats.
  • a motion sensor halted the stimulus and timer.
  • a maximal cut-off of 40 seconds was utilized to prevent tissue damage.
  • Paw withdrawal latencies were thus determined to the nearest 0.1 seconds.
  • Reversal of thermal hyperalgesia was indicated by a return of the paw withdrawal latencies to the pre-tremeant baseline latencies (i.e., 21 seconds).
  • Antinociception was indicated by a significant (p ⁇ 0.05) increase in paw withdrawal latency above this baseline. Data were converted to % antihyperalgesia or % antinociception by the formula:
  • a preferred combination will result, for example, in the same amount of pain relief after administering 50 mg of an NSAID or opioid in combination with 50 mg of a synthetic superoxide dismutase catalyst as would normally result from administering 500 mg of an NSAID or opioid alone or 500 mg of a synthetic superoxide dismutase catalyst alone.
  • the preferred combinations of the present invention treat pain to a greater extent when compared to treating pain with an analgesic alone or a synthetic superoxide dismutase catalyst alone.
  • a preferred combination will result, for example, in an equivalent amount of pain relief after administering 500 mg of an NSAID or opioid in combination with 50 mg of a synthetic superoxide dismutase catalyst as would normally result from administering 1 ,000 mg of the NSAID or opioid or 1 ,000 mg of a synthetic superoxide dismutase catalyst alone.
  • preferred combinations result in additive or synergistic antinociceptive effects allowing an NSAID or opioid to be administered in a dosage that is at least 50% less than the same NSAID or opioid administered alone. More preferably, the NSAID or opioid combination may be administered in a dosage that is at least 25% less than the same NSAID or opioid administered alone to achieve said therapeutic effect.
  • the NSAID or opioid may be administered in a dosage that is at least 10% less than the same NSAID or opioid administered alone to achieve said therapeutic effect. And still more preferably, the NSAID or opioid may be administered in a dosage that is at least 1% less than the same NSAID or opioid administered alone to achieve said therapeutic effect.
  • Example 4 - SC-72325A Treats Pain Analgesic effects provided by subcutaneous injection of SC-72325A was studied by formalin-induced hind paw licking response.
  • Male CD-1 mice (Charles River, 28-35 gm) were allowed to feed ad libitum. Mice were housed 5-7 per cage in a temperature controlled room with a twelve hour light-dark cycle. Determination of antinociception was assessed between 7:00 and 10:00 AM. Groups consisted of 7-14 mice, and each animal was used for one experimental condition. The antinociceptive effects of SC-72325A were tested in the formalin-induced hind paw licking procedure (Hunskaar et al., Pain, 30: 103-114, 1987).
  • mice were injected with by sub-plantar administration with formalin (20 ⁇ g of a 1% stock solution) and the duration of paw licking was monitored in the periods of 5-10 minutes (Phase I) and 15-30 minutes (Phase II) thereafter.
  • SC-72325A (10 mg/kg) was given s.c. 10 minutes prior to formalin. At 10 mg/kg, the s.c. injection of SC-72325A had a small inhibitory effect on phase 1 of the response but nearly completely abolished Phase II of the response.
  • Example 5 Antihyperalgesia and Antinociception Synergy using SC-72325A and Morphine Combination
  • Carrageenan-induced inflammation is a well characterized and commonly employed model of peripheral inflammation. This procedure reliably produces a marked inflammatory response within 3 hours of injection which is indicated by swelling of the hind paw, edema, rubor and hyperalgesia and allodynia.
  • Peripheral inflammation was induced in the hind paw of male Sprague-Dawley rats by injecting 0.1 ml of a 2% /.-carrageenan suspension into the subplanar surface of the hind paw of lightly ether-anesthetized rats. Testing was performed 15, 30, 45, 60, 120 and 180 minutes after drug injections.
  • the s.c. injection of SC-72325A produced time-related and dose-dependant antihyperalgesia over the dose range of 0.3 to 30 mg/kg.
  • morphine also produced time-related and dose-dependent antihyperalgesia and antinociception over the dose range of 0.03 to 10 mg/kg.
  • the 1 :1 combination of SC-72325A with morphine produced antihyperalgesia activities at much lower doses than either drug alone. Isobolographic analyses revealed that the combination of SC-72325A with morphine resulted in a definitive synergistic interaction against hyperalgesia; the A 50 values with confidence intervals are presented in Table 1 , below.
  • the antihyperalgesic A 50 value for the 1 :1 combination of SC-72325A plus morphine was 0.046 mg/kg, s.c, which was significantly (p ⁇ 0.05) less than the calculated theoretical A 50 value for the combination if the activity was additive. See Table 1. SC-72325A also exhibited a slight antinociceptive effect. TABLE 1 Antihyperalgesia A 50 (mg/kg, s.c.) SC-72325A 1.34 Morphine 0.22 Morphine + SC-72325A 0.046 Theoretical Additive Curve 0.380
  • Example 6 Formulations of Compositions The following formulations are provided as examples of composition preparations according to the present invention:
  • Hard gelatin capsules are prepared using the following ingredients: TABLE 2 Ingredients (mg/capsule) Active Ingredient 250.0 Starch 305.0 Magnesium stearate 5.0
  • the above ingredients are mixed and filled into hard gelatin capsules in 560 mg quantities.
  • Formulation 2 A tablet formula is prepared using the following ingredients: TABLE 3 Ingredients (mg/tablet) Active Ingredient 250.0 Cellulose, microcrystalline 400.0 Colloidal silicon dioxide 10.0 Stearic acid 5.0
  • the components are blended and compressed to form tablets, each weighing 665 mg.
  • Formulation 3 A dry powder inhaler formulation is prepared containing the following components: TABLE 4 Ingredients Weight % Active ingredient 5 Lactose 95
  • the active ingredient is mixed with the lactose and the mixture is added to a dry powder inhaling appliance.
  • Formulation 4 Tablets each containing 60 mg of active ingredient, are prepared as follows: TABLE 5 Ingredients milligrams Active ingredient 60.0 Starch 45.0 Microcrystalline cellulose 35.0 Polyvinylpyrrolidone (as 10% solution in water) 4.0 Sodium carboxymethyl starch 4.5 Magnesium stearate 0.5 Talc 1.0 Total 150.0 The active ingredient, starch and cellulose are passed through a No. 20 mesh U.S. sieve and mixed thoroughly. The solution of polyvinylpyrrolidone is mixed with the resultant powders which are then passed through a 16 mesh U.S. sieve. The granules as produced are dried at 50-60 °C and passed through a 16 mesh U.S. sieve.
  • the sodium carboxymethyl starch, magnesium stearate, and talc previously passed through a No. 30 mesh U.S. sieve, are then added to the granules which, after mixing, are compressed on a tablet machine to yield tablets each weighing 150 mg.
  • Formulation 5 Capsules, each containing 80 mg of active ingredient are made as follows: TABLE 6 Ingredients milligrams Active ingredient 80.0 Starch 109.0 Magnesium stearate 1.0 Total 190.0
  • the active ingredient, cellulose, starch, and magnesium stearate are blended, passed through a No. 20 mesh U.S. sieve, and filled into hard gelatin capsules in 190 mg quantities.
  • Formulation 6 Suppositories, each containing 225 mg of active ingredient, are made as follows: TABLE 7 Ingredients milligrams Active Ingredient 225 Saturated fatty acid glycerides to 2000
  • the active ingredient is passed through a No. 60 mesh U.S. sieve and suspended in the saturated fatty acid glycerides previously melted using the minimum heat necessary. The mixture is then poured into a suppository mold of nominal 2.0 g capacity and allowed to cool.
  • Formulation 7 Suspensions, each containing 50 mg of active ingredient per 5.0 ml dose are made as follows: TABLE 8 Ingredients Active ingredient 50.0 mg Xanthan gum 4.0 mg Sodium carboxymethyl cellulose (11%) Microcrystalline cellulose (89%) 50.0 mg Sucrose 1.75 g Sodium benzoate 10.0 mg Flavor q.v. Color q.v. Purified water to 5.0 ml
  • the active ingredient, sucrose and xanthan gum are blended, passed through a No. 10 mesh U.S. sieve, and mixed with a previously made solution of the microcrystalline cellulose and sodium carboxymethyl cellulose in water.
  • the sodium benzoate, flavor, and color are diluted with some of the water and added with stirring. Sufficient water is then added to produce the required volume.
  • Formulation 8 Capsules, each containing 150 mg of active ingredient, are made as follows: TABLE 9 Ingredients milligrams Active ingredient 150.0 Starch 407.0 Magnesium stearate 3.0 Total 560.0
  • the active ingredient, cellulose, starch, and magnesium stearate are blended, passed through a No. 20 mesh U.S. sieve, and filled into hard gelatin capsules in 560 mg quantities.

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Abstract

L'invention concerne une méthode permettant de fournir un service consistant à identifier un inhibiteur de nitration qui permet de traiter, prévenir ou inhiber la douleur et de préparer une forme posologique thérapeutiquement efficace dudit inhibiteur de nitration. L'invention concerne également une méthode qui permet de générer un revenu.
PCT/US2004/036186 2003-10-31 2004-10-31 Procedes permettant de generer ou d'augmenter des revenus lies au commerce des inhibiteurs de la douleur Ceased WO2005041886A2 (fr)

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US8791063B2 (en) 2011-08-25 2014-07-29 Hoffmann-La Roche, Inc. Shortened tetranectin-apolipoprotein A-I fusion protein, a lipid particle containing it, and uses thereof
US8987245B2 (en) 2008-04-02 2015-03-24 Jonathan R. Brestoff Parker Composition and method for affecting obesity and related conditions
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US8598150B1 (en) 2008-04-02 2013-12-03 Jonathan R. Brestoff Composition and method for affecting obesity and related conditions
US8809312B2 (en) 2008-04-02 2014-08-19 Jonathan R. Brestoff Composition and method for affecting obesity and related conditions
US8987245B2 (en) 2008-04-02 2015-03-24 Jonathan R. Brestoff Parker Composition and method for affecting obesity and related conditions
US8791063B2 (en) 2011-08-25 2014-07-29 Hoffmann-La Roche, Inc. Shortened tetranectin-apolipoprotein A-I fusion protein, a lipid particle containing it, and uses thereof
US9139640B2 (en) 2011-08-25 2015-09-22 Hoffmann-La Roche Inc. Shortened tetranectin-apolipoprotein A-1 fusion protein, a lipid particle containing it, and uses thereof
US9757395B2 (en) 2012-12-20 2017-09-12 Otitopic Inc. Dry powder inhaler and methods of use
US9757529B2 (en) 2012-12-20 2017-09-12 Otitopic Inc. Dry powder inhaler and methods of use
US12329899B2 (en) 2012-12-20 2025-06-17 Aspeya US Inc. Dry powder inhaler and methods of use
US10149823B2 (en) 2013-04-30 2018-12-11 Otitopic Inc. Dry powder formulations and methods of use
US11819569B2 (en) 2013-04-30 2023-11-21 Vectura Inc. Treating inflammation with inhaled aspirin
US11865210B2 (en) 2013-04-30 2024-01-09 Vectura Inc. Dry powder formulations and methods of use
US10195147B1 (en) 2017-09-22 2019-02-05 Otitopic Inc. Dry powder compositions with magnesium stearate
US10786456B2 (en) 2017-09-22 2020-09-29 Otitopic Inc. Inhaled aspirin and magnesium to treat inflammation
US11077058B2 (en) 2017-09-22 2021-08-03 Otitopic Inc. Dry powder compositions with magnesium stearate

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