Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • 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
• • 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
  • A61K31/13—Amines
  • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
  • A61K31/445—Non condensed piperidines, e.g. piperocaine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/47—Quinolines; Isoquinolines
  • A61K31/485—Morphinan derivatives, e.g. morphine, codeine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/20—Pills, tablets, discs, rods
  • A61K9/2004—Excipients; Inactive ingredients
  • A61K9/2013—Organic compounds, e.g. phospholipids, fats
  • A61K9/2018—Sugars, or sugar alcohols, e.g. lactose, mannitol; Derivatives thereof, e.g. polysorbates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/20—Pills, tablets, discs, rods
  • A61K9/2004—Excipients; Inactive ingredients
  • A61K9/2022—Organic macromolecular compounds
  • A61K9/205—Polysaccharides, e.g. alginate, gums; Cyclodextrin
  • A61K9/2054—Cellulose; Cellulose derivatives, e.g. hydroxypropyl methylcellulose
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
  • A61K9/4841—Filling excipients; Inactive ingredients
  • A61K9/4858—Organic compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
  • A61K9/4841—Filling excipients; Inactive ingredients
  • A61K9/4866—Organic macromolecular compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
  • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
  • A61K9/5005—Wall or coating material
  • A61K9/5021—Organic macromolecular compounds
  • A61K9/5036—Polysaccharides, e.g. gums, alginate; Cyclodextrin
  • A61K9/5042—Cellulose; Cellulose derivatives, e.g. phthalate or acetate succinate esters of hydroxypropyl methylcellulose
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
  • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
  • A61K9/5084—Mixtures of one or more drugs in different galenical forms, at least one of which being granules, microcapsules or (coated) microparticles according to A61K9/16 or A61K9/50, e.g. for obtaining a specific release pattern or for combining different drugs
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/10—Laxatives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/04—Centrally acting analgesics, e.g. opioids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/26—Psychostimulants, e.g. nicotine, cocaine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• ABC proteins play a central role in living cells through their role in nutrient uptake, protein, drug and antibiotic secretion, osmoregulation, antigen presentation, signal transduction and others.
• the majority of ABC proteins have a translocation function either in import of substrates or secretion of cellular products or xenobiotics.
• ABC ATP-binding cassette
• the structure and function of drug transporters have been extensively reviewed, including by Benet, et al, J. Control. Rel. 39:139-143 (1996) for the gut barrier, by Chiou, et al, Pharm. Res. 17(8):903-905 (2000) for the liver barrier (and includes gut barrier references), and by Tduji, et al, in Adv. Drug Deliv. Rev. 36:277-290 (1999) for the blood-brain barrier.
• the family of drug transporters includes two different subfamilies, the multidrug resistance (MDR) proteins, such as PGP, and the multidrug resistance-associate protein (MRP) family.
• MDR multidrug resistance
• MRP multidrug resistance-associate protein
• the human multidrug resistance-associated protein family currently has seven members (Borst et al, J. Natl Cancer Inst. 92:1295-
• MDR1 multi- drug resistance protein
• PGP P-glycoprotein
• P-glycoprotein is an ATP-dependent drug transporter that is predominantly found in the apical membranes of a number of epithelial cell types in the body, including the luminal membrane of the brain capillary endothelial cells that make up the blood-brain barrier. Expression of PGP, localized to cell membranes may affect the bioavailability of drug molecules that are substrates for this transporter. Knockout mice lacking the gene encoding P-glycoprotein show elevated brain concentrations of multiple systemically administered drugs, including opioids as wells as chemotherapeutic agents. Chen and Pollack, J. Pharm. Exp. Ther.
• MRP is able to transport metallic oxyanions and glutathione and other conjugates, including peptidyl leukotrienes.
• Agents that inhibit organic ion transport, such as probenecid, can block MRP activity.
• the blood brain barrier is a capillary barrier comprising a continuous layer of tightly bound endothelial cells.
• the interendothelial junctions between the cells of the blood brain barrier act to keep potentially noxious substances away from the brain.
• the continuity produced by the tight junctions between individual cells of the blood brain barrier enables the cerebrocapillary endothelium to act like a plasma membrane.
• Small molecules m.w. ⁇ 200 daltons
• larger substances are substantially excluded. This protects the brain microenvironment from rapid changes in the composition of the blood. Numerous pharmaceutical substances have their pharmacological action in the central nervous system.
• delivering these pharmaceutical substances to their active sites in the central nervous system (CNS), particularly the brain can be problematic due to the very limited permeability of the blood brain barrier, which discourages transport of many therapeutically active agents into the brain.
• the blood brain barrier may actively export molecules that cross the barrier, e.g., by leakage through the tight junctions between the endothelial cells or by non-specific passive diffusion across the endothelial membrane. After the compounds enter the brain, active efflux of these compound from the apical surface of the endothelial cells would place the compounds back into the endothelial cells and thus back into the blood. Such a mechanism would effectively decrease the cerebral concentration of these compounds.
• CNS-active agents One important class of CNS-active agents is the class of opioid compounds.
• Opioid receptor agonists including morphine sulfate (hereafter called morphine or MS), have been marketed for many years and are widely used for the relief of moderate to severe acute and chronic pain.
• An opioid receptor agonist such as morphine, exerts its primary effects on the central nervous system and organs containing smooth muscle, and acts as an agonist interacting with stereospecific and saturable binding sites or receptors in the brain, spinal cord, and other tissues.
• the principal therapeutic actions are analgesia and sedation.
• Opioid receptor antagonists are generally accepted for use in the treatment of human conditions of ailments for reversing opioid toxicity and overdoses, and in preventing abuse of opioid receptor agonists, such as heroin or morphine.
• opioid receptor agonists such as heroin or morphine.
• the antagonists such as naloxone or naltrexone is used in relatively high concentrations in order to effectively block the activity and/or effects of the opioid receptor agonist by antagonizing the opioid receptor agonist at opioid receptors on nociceptive neurons.
• the ability of the blood brain barrier to protect the nervous system from exogenous substances has impeded the development of therapies for a wide variety of disorders and conditions of the central nervous system.
• the blood brain barrier presents a particularly difficult obstacle to treating conditions in which the therapeutic agents must act upon sites within the central nervous system, particularly the brain.
• SUMMARY OF THE INVENTION The present invention is directed to novel methods and compositions with drug transporter inhibitors.
• Such inhibitors according to the invention modulate the activity of ABC transporter proteins and include inhibitors of MDR proteins, such as PGP, as well as MRP proteins.
• Such methods and compositions are designed to achieve, for example, enhanced efficacy of opioid and/or non-opioid CNS-active agents, prevention and/or reversal of tolerance to, dependence upon or withdrawal from opioid and/or non-opioid CNS-active agents, as well as improved treatment of chronic pain patients.
• the present invention is based in part on surprising results from transport studies of drug agents across the blood brain barrier that demonstrate that compounds of a defined structure according to the invention, including naltrexone, nalmafene and naloxone, are inhibitors of ABC transporter proteins, such as PGP, and unexpectedly increase the concentration in the brain of CNS-active agents, including opioid receptor agonists such as morphine and oxycodone. Also unexpectedly demonstrated is the reduction of efflux of such CNS-active agents from the brain by inhibitors of ABC transporter proteins according to the invention.
• the present invention provides a novel class of drug transporter inhibitors that act by inhibiting ABC transporter proteins and further provides a pharmacophore that allows the identification of new drug targets that are inhibitors of ABC transporter proteins. Also provided are new methods of screening for and/or identifying compounds that inhibit the transport (e.g., efflux or influx) of CNS-active agents across the blood brain barrier.
• ABC transporter inhibitors identified according to the invention increase brain concentrations of CNS-active agents. Such inhibitors increase the influx into the brain and/or or reduce the efflux from the brain of such CNS-active agents.
• the present invention provides methods and compositions for enhancing the efficacy of a non-opioid CNS-active agent by co-administering to a patient a therapeutic or sub-therapeutic dose of the non-opioid CNS-active agent and an amount of an inhibitor of a drug transporter effective to reduce efflux of the non-opioid CNS-active agent from the brain and/or to increase the concentration of the non-opioid CNS-active agent in the brain, where the drug transporter is an ABC drug transporter.
• the present invention also methods and compositions for enhancing the efficacy of an opioid CNS-active agent by co-administering a therapeutic or sub-therapeutic dose of the opioid CNS-active agent with a non-opioid drug transporter inhibitor, such that the amount of non-opioid drug transporter inhibitor is effective to reduce efflux of the opioid CNS- active agent from the brain and/or to increase the concentration of the opioid CNS-active agent in the brain.
• the present invention further provides methods and compositions for reversing or preventing tolerance to CNS-active agent, including an opioid CNS-active agent, by administering a drug transporter inhibitor to a patient, including a patient who is tolerant to the CNS-active agent, such that the amount of drug transporter inhibitor administered is sufficient to decrease efflux of the CNS-active agent form the brain and/or to increase the concentration of the CNS-active agent in the brain.
• the present invention also provides methods of treating a patient experiencing chronic pain by co-administering to a patient a therapeutic or sub-therapeutic dose of an CNS-active agent, including an opioid CNS-active agent, and an amount of a drug transporter inhibitor effective to increase the concentration of the CNS-active agent in the brain.
• the co-administration may be repeated over a period of time that is greater than the period of time in which the patient would develop tolerance to or develop a dependence upon the CNS-active agent administered in the absence of the drug transporter inhibitor.
• the invention also provides methods of controlling chronic pain without tolerance, dependence and/or withdrawal by co-administering a therapeutic or sub-therapeutic dose of a CNS-active agent, including an opioid CNS-active agent, and an amount of a drug transporter inhibitor effective to decrease efflux of the CNS-active agent form the brain and/or increase the concentration of CNS-active agent, including an opioid CNS-active agent, in the brain.
• the present invention further provides methods and composition for enhancing the efficacy of a non-opioid CNS-active agent by co-administering non-opioid CNS-active agent with an opioid receptor antagonist, such that the amount of antagonist is effective to reduce efflux of the agent from the brain and/or increase the concentration of the agent in the brain.
• BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 illustrates the chemical structures of naltrexone, naloxone, nalmefene, 6- ⁇ - naltrexol and nalorphine.
• Fig. 2 presents an overlay of the opioid analogues, naltrexone, naloxone, nalmefene, 6- ⁇ - naltrexol and nalorphine.
• Fig. 3 A shows the molecular orbitals and electrostatic potential of nalmefene as calculated using Spartan (Wavefunction, Inc.).
• FIG. 3B shows the molecular orbitals and electrostatic potential of naloxone as calculated using Spartan (Wavefunction, Inc.).
• FIG. 4A-HH provide the 200 nearest neighbors of opioid analogues examined in the
• FIG. 5 illustrates the effect of naltrexone on the concentration of morphine in the brain of male and female rats.
• FIG. 6 illustrates that administration of naltrexone to morphine-tolerant mice breaks tolerance.
• FIG. 7 illustrates that administration of naltrexone in combination with morphine prevents the mice from developing a tolerance to morphine
• the present invention is based in part on surprising results from transport studies of drug agents across the blood brain barrier that demonstrates that compounds previously identified as opioid receptor antagonists are inhibitors of ABC drug transporter proteins, including of the P-glycoprotein found at the blood brain barrier, PGP la.
• opioid receptor antagonists such as naloxone, nalmefene and naltrexone
• Such antagonists also unexpectedly reduced the efflux and/or increased the influx of the co-administered agents.
• the present invention provides a novel class of drug transporter inhibitors that act by inhibiting ABC transporter proteins and their associated ATPase as described herein and further provides a pharmacophore that identifies new drug targets that are inhibitors of ABC transporter proteins.
• transporter and “drug transporter” refer to a protein for the carrier-mediated influx and efflux of drugs and endocytosis of biologically active molecules, including across a gut, liver, or blood-brain barrier.
• An inhibitor of a transporter is expected to increase the bioavailability of an active agent according to the invention, wherein the transporter inhibitor reduces efflux across the blood-brain barrier, or the cellular membrane of a cancerous or microbial cell, thereby enhancing the therapeutic effectiveness of the active agent.
• the drug transporter protein is a member of the ABC superfamily.
• the drug transporter may either be a multidrug resistance protein (MDR) or a multidrug resistance-associated protein (MRP).
• MDR proteins multidrug resistance protein
• MRP multidrug resistance-associated protein
• the predominant difference between MDR proteins and MRPs is that MRPs require glutathione, in addition to ATP, in order to transport compounds across a biological barrier.
• the range of substrates may vary from one drug transporter protein to another.
• ABC transporter inhibitors identified according to the invention can increase brain concentrations of co-administered agents that are substrates of the transporters.
• the WalkerA region the WalkerB region
• a short consensus sequence leucine-serine-glycine-glycine-glutamine, or LSGGQ.
• the short consensus sequence LSGGQ is found in essentially every known ABC protein.
• the QS AR analysis of the present invention provides the very surprising result that the opioid receptor antagonists that act as PGP inhibitors bind to this LSGGQ consensus sequence.
• the present invention defines a strictly conserved inhibition site shared among all ABC drug transporter proteins. Therefore, the opioid receptor antagonists of the present invention will function as an inhibitor of every ABC drug transporter protein that shares the LSGGQ conserved sequence.
• inhibitor of a drug transporter or “drug transporter inhibitor” refers to a compound that binds to a drug transporter protein and inhibits, i.e., either completely blocks or merely slows, transport of compounds across biological barriers.
• ABC drug transporter inhibitor refers to an inhibitor of one or more of the proteins in the ABC superfamily of drug transporters. Drugs that inhibit drug transporters can alter the absorption, disposition and elimination of co-administered drugs and can enhance bioavailability or cause unwanted drug-drug interactions. Interaction with drug transporters can be studied using either direct assays of drug transport in polarized cell systems or with indirect assays such as drug-stimulated ATPase activity and inhibition of the transport of fluorescent substrates.
• Drugs affected by the drug transporter, P-glycoprotein, at the blood- brain barrier include ondasetron, dexamethasone, domperidone, loperamide, doxorubicin, neifinavir, indinevir, sugguinavir, erythromycin, digoxin, vinblastine, paclitaxel, invermectin and cyclosporm.
• Known inhibitors of P-glycoprotein include ketoconazole, verapamil, quinidine, cyclosporin, digoxin, erythromycin and loperamide. See, e.g., Intl. J. Clin. Pharmacol. Ther. 38:69-74 (1999).
• opioid receptor antagonists such as naloxone, naltrexone and nalmefene, as potent inhibitors of ABC drug transporters, such as P-glycoprotein.
• opioid receptor antagonist is an opioid compound or composition including any active metabolite of such compound or composition that in a sufficient amount attenuates (e.g., blocks, inhibits, prevents or competes with) the action of an opioid receptor agonist.
• An opioid receptor antagonist binds to and blocks (e.g., inhibits) opioid receptors on nociceptive neurons.
• Opioid receptor antagonists include: naltrexone (marketed in 50mg dosage forms as ReNia® or Trexan®), nalaxone (marketed as ⁇ arcan®), nalmefene, methylnaltrexone, naloxone, methiodide, nalorphine, naloxonazine, nalide, nalmexone, nalbuphine, nalorphine dinicotinate, naltrindole ( ⁇ TI), naltrindole isothiocyanate ( ⁇ TII), naltriben ( ⁇ TB), nor-binaltorphimine (nor-B ⁇ I), b-funaltrexamine (b-F ⁇ A), B ⁇ TX, cyprodime, ICI-174,864, LY117413, MR2266, or an opioid receptor antagonist having the same pentacyclic nucleus as nelmefene, naltrexone,
• the present invention contemplates enhancing the efficacy of non- opioid C ⁇ S-active agents by co-administering the C ⁇ S-active agent with an ABC drug transporter inhibitor, including an opioid transporter inhibitor, such as an opioid receptor antagonist.
• an opioid transporter inhibitor such as an opioid receptor antagonist.
• the opioid receptor antagonists, naltrexone, naloxone and nalmefene, are particularly suited for the present invention.
• the present invention also contemplates enhancing the efficacy of opioid C ⁇ S-active agents, such as an opioid receptor agonist, by co-administering the opioid C ⁇ S-active agent, with a non-opioid ABC drug transporter inhibitor.
• some inhibitors of PGP are known in the art, many of these are extremely toxic, especially if used repeatedly over a period of time.
• ketoconazole when used orally, ketoconazole has been associated with hepatic toxicity, including some fatalities.
• the opioid receptor antagonists historically have limited side effects, particularly at the low concentrations administered in the present invention.
• Each of the antagonists naltrexone, naloxone and nalmefene have been approved by the FDA for use in antagonistically effect amounts for treatment of opioid overdose and addiction.
• a quantitative structure-activity relationship (QSAR) analysis of several opioid drug transporter inhibitors of the present invention defines a pharmacophore consisting of two essential hydroxyls (at positions 3 and 14), a nitrogen with an appended hydro-phobic region, and electron density at the 6-position of the opioid compounds.
• QSAR quantitative structure-activity relationship
• R 1 is CH 2 or O; wherein R 2 is a cycloalkyl, unsubstituted aromatic, alkyl or alkenyl; and wherein R 3 is O, CH 2 or NH.
• the ABC drug transporter inhibitors including opioid receptor antagonists according to the invention may be co-administered with any non-opioid CNS-active agent.
• opioid CNS-active agents including opioid receptor agonists
• opioid receptor agonists may be co- administered with non-opioid ABC drug transporter inhibitors according to the invention.
• Opioid receptor agonists may be additionally administered with the co-administered CNS- active agents and the ABC drug transporter inhibitors.
• co-administer “co- administration,” “concurrent administration” and “co-treatment” refer to administration of an active agent and a drug transporter inhibitor, in conjunction or combination, together, or before or after each other.
• the active agent and the drug transporter inhibitor may be administered by different routes.
• the active agent may be administered orally and the drug transporter inhibitor intravenously, or vice versa.
• the active agent and the drug transporter inhibitor are preferably both administered orally, as immediate or sustained release formulations.
• the active agent and drug transporter inhibitor may be administered simultaneously or sequentially, as long as they are given in a manner to allow both agents to achieve effective concentrations to yield their desired therapeutic effects.
• CNS-active agent means any therapeutic agent that acts at a site within the central nervous system (CNS), especially vv ⁇ thin the brain.
• CNS-active agents include (1) general CNS depressants, such as, anesthetic gases and vapors, aliphatic alcohols, and some hypnotic-sedative drugs; (2) general CNS stimulants, such as pentylenetetrazol, and the methylxanthines; and (3) drugs that selectively modify CNS function, such as anticonvulsants, antiparkinsonism drugs, opioid and non-opioid analgesics, appetite suppressants, antiemetics, analgesic-antipyretics, certain stimulants, antidepressants, antimanic agents, antipsychotic agents, sedatives and hypnotics.
• general CNS depressants such as, anesthetic gases and vapors, aliphatic alcohols, and some hypnotic-sedative drugs
• general CNS stimulants such as pentylenetetrazol, and
• CNS-active agents are not limited to agents that act solely within the central nervous system.
• CNS-active agents are opioid receptor agonists, such as morphine or oxycodone, which binds to opioid receptors on nociceptive neurons.
• non- opioid CNS-active agents include, but are not limited to, valium, lithium, halcyon and ambien.
• an ABC drug transporter inhibitor such as opioid receptor antagonist
• an opioid receptor antagonist an ABC drug transporter inhibitor that is necessary to increase the concentration of a co-administered CNS-active agent in the brain will vary from individual to individual.
• the amount of the inhibitor for example, an opioid receptor antagonist, necessary to achieve the desired effect will also vary from one antagonist to the next. This amount is readily determinable by one skilled in the art according to the invention.
• the ABC drug transporter inhibitor including an opioid inhibitor such as an the opioid receptor antagonist or a non-opioid inhibitor, may be administered with a therapeutically effective amount of the CNS-active agent.
• “Therapeutic effect” or “therapeutically effective” refers to an effect or effectiveness that is desirable and that is an intended effect associated with the administration of an active agent according to the invention.
• the therapeutic effect of a CNS-active agent that is an opioid receptor agonist would include analgesia or pain relief or feeling good or calming so as to reduce heart rate, blood pressure or breathing rate.
• a “therapeutic amount” is the amount of an active agent sufficient to provide a therapeutic effect.
• the ABC drug transporter inhibitor such as an opioid receptor antagonist
• a "sub-therapeutic amount” is an amount of the active agent that does not cause a therapeutic effect in a patient administered the active agent alone, but when used in combination with the opioid or non-opioid drug transporter inhibitor is therapeutically effective.
• Co- administering a sub-therapeutic dose of the active agent with the ABC drug transporter inhibitor, such as an opioid receptor antagonist has many clinical advantages. By administering a smaller amount of the therapeutic agent, it will be possible to obtain the same brain concentration of the active agent while providing a much lower total systemic concentration. This effect will result in fewer system side effects.
• An "adverse side effect" of an opioid agonist is a side effect in humans, typically associated with opioid analgesics such as morphine, including nausea vomiting, dizziness, somnolence/sedation, pruritus, reduced gastrointestinal motility including constipation, difficulty in urination, peripheral vasodilation including leading to orthostatic hypotension, headache, dry mouth, sweating, asthenia, dependence, mood changes (e.g., dysphoria, euphoria), or lightheadedness.
• An “adverse side effect” also includes a serious adverse side effect such as respiratory depression or also apnea, respiratory arrest, circulatory depression, hypotension or shock.
• opioid agonists have been documented to produce numerous adverse side effects.
• side effects that have been recognized for products containing morphine or other opioid agonists are: respiratory depression; depression of the cough reflex; miosis; reduced gastrointestinal motility including constipation; peripheral vasodilation which may result in orthostatic hypotension; and release of histamine.
• Adverse side effects that are of particular interest in human subjects include nausea, vomiting, dizziness, headache, somnolence (sedation), and pruritus.
• PDR Physician Desk Reference
• morphine respiratory depression; apnea; circulatory depression; shock respiratory arrest, and cardiac arrest
• oxycodone light-headedness, euphoria, dysphoria, constipation, skin rash
• hydrocodone mental clouding, lethargy, impairment of mental and physical performance, anxiety, fear, dysphoria, dependence, mood changes; constipation; ureteral spasm; spasm of vesical sphincter and urinary retention
• tramadol seizures; anaphylactoid reactions (lessened resistance to toxins); asthenia; sweating; dyspepsia; dry mouth; diarrhea
• CNS stimulation (“CNS stimulation” is a composite that can include nervousness, anxiety, agitation, tremor, spasticity, euphoria, emotional liability and hallucinations); malaise; vasodilation; anxiety, confusion, coordination disturbance,
• the invention is based in part upon corresponding relationship between drug transporter protein function and the concentration of the opioid agent in the central nervous system, particularly in the brain. Without being limited to a particular theory, it is believed that the increase in brain concentrations is mediated by inhibition of active transport of the CNS-active agents by P-glycoprotein.
• the agents cross the blood brain barrier according to normal physiological paths, e.g., diffusion of lipophilic molecules across the cell membrane of the endothelial cells lining the cerebral capillaries. Once across the blood brain barrier, the agent is captured by the drug transporter protein and swept back to the exterior side of the blood brain barrier.
• the active efflux of therapeutic agents results in an artificially low concentration of the agent within the central nervous system, particularly in the brain.
• some drug transporter inhibitors such as nalmefene and naltrexone additionally inhibit the ATPase activity of an ABC transporter protein and thereby may also increase influx of drugs through the ABC proteins transmembrane channel. Accordingly, increased brain concentrations of CNS-active agents that are ABC protein substrates may be achieved either through inhibiting active efflux by the ABC protein, or through increasing influx, for example, by inhibiting the associated ATPase and thus allowing passage through the ABC protein, or by a combination of both decreasing efflux and increasing influx.
• an opioid CNS- active agent such as morphine
• a drug transporter inhibitor such as naltrexone
• co-administration of an opioid CNS-active agent, such as morphine, and a drug transporter inhibitor, such as naltrexone results in a higher concentration of mo hine in the brain as compared to that found in a subject who received morphine alone.
• a drug transporter inhibitor e.g., a non-opioid inhibitor
• opioid refers to compounds or compositions including metabolites of such compounds or compositions which bind to specific opioid receptors and have agonist (activation) or antagonist (inactivation) effects at these receptors, and thus are “opioid receptor agonists" or “opioid receptor antagonists.”
• opioid alkaloids such as the agonist morphine and its metabolite morphine-6-glucuronide and the antagonist naltrexone and its metabolite and opioid peptides, including enkephalins, dynorphins and endorphins.
• the opioid can be present as a member selected from an opioid base and an opioid pharmaceutically acceptable salt.
• the pharmaceutically acceptable salt embraces an inorganic or an organic salt.
• Representative salts include hydrobromide, hydrochloride, mutate, succinate, n-oxide, sulfate, malonate, acetate, phosphate dibasic, phosphate monobasic, acetate trihydrate, bi(heplafluorobutyrate), maleate, bi(methylcarbamate), bi(pentafluoropropionate), mesylate, bi(pyridine-3-carboxylate), bi(trifluoroacetate), bitartrate, chlorhydrate, fumarate and sulfate pentahydrate.
• opioid refers to drugs derived from opium or related analogs.
• a "non-opioid CNS-active agent” is a CNS- active agent, as defined above, that does not bind to specific opioid receptors or if it binds one that fails to activate or inactivate the receptor.
• opioid CNS-active agents are opioid receptor agonists.
• An "opioid receptor agonist” is an opioid compound or composition including any active metabolite of such compound or composition that binds to and activates opioid receptors on nociceptive neurons, which mediate pain.
• opioid receptor agonists have analgesic activity (with measurable onset, peak, duration and/or total effect) and can product analgesia.
• Opioid receptor agonists include: alfentanil, allylprodine, alphaprodine, anileridine, apomorphine, apocodeine, benzylmorphine, bezitramide, buprenorphine, butorphanol, clonitazene, codeine, cyclazocine, cyclorphen, cyprenorphine, desomorphine, dextromoramide, dezocine, diampromide, dihydrocodeine, dihyrdomorphine, dimenoxadol, dimepheptanol, dimethylthiambutene, dioxyaphetyl butyrate, dipipanone, eptazocine, ethoheptazine, ethylmethylthiambutene, ethylmorphine, etonitazene, fentanyl, heroin, hydrocodone, hydroxymethylmorphinan, hydromorphone, hydroxype
• Preferred opioid receptor agonists for human use include morphine, hydrocodone, oxycodone, codeine, fentanyl (and its relatives), hydromorphone, meperidine, methadone, oxymorphone, propoxyphene or tramadol, or mixtures thereof. Particularly preferred agonists include morphine, oxycodone, hydrocodone or tramadol.
• Opioid receptor agonists include exogenous or endogenous opioids. As a compound that activates opioid receptors on nociceptive neurons, opioid receptor agonists are commonly used as analgesic agents.
• An “Analgesia” refers to the attenuation, reduction or absence of sensibility to pain, including the provision of pain relief, the enhancement of pain relief, or the attenuation of pain intensity.
• An “analgesic” amount refers to an amount of the opioid receptor agonist which causes analgesia in a subject administered the opioid receptor agonist alone, and includes standard doses of the agonist which are typically administered to cause analgesia (i.e., mg doses).
• an “analgesic” amount refers to an amount that results in analgesic efficacy, for example, as measured by a subject with a pain relief score or a pain intensity difference score, at a given timepoint, or over time, or as compared to a baseline, and includes calculations based on area under the curve (AUC) such as TOTPAR or SPID from such pain relief scores or pain intensity difference scores.
• a "hypo-analgesic” amount is a less-than-analgesic amount, including an amount which is not analgesic or is weakly analgesic in a subject administered the opioid receptor agonist alone, and further includes an "anti-analgesic” or “algesic” amount which is an amount which increases pain.
• a “sub-analgesic” amount is an amount that does not cause analgesia in a subject administered the opioid receptor agonist alone, but when used in combination with the opioid receptor antagonist, results in analgesia.
• the pharmaceutical compositions or dosage forms of this invention may be utilized in compositions such as capsules, tablets or pills for oral administration, suppositories for rectal administration, liquid compositions for parenteral administration and the like.
• compositions or dosage forms of this invention may be used in the form of a pharmaceutical preparation, for example, in solid or semisolid form, which contains one or more of the drug transporter inhibitors, as an active ingredient, alone, or in combination with one or more therapeutic agents.
• Any drug transporter inhibitor or therapeutic agent may be in admixture with an organic or inorganic carrier or excipient suitable for external, enteral or parenteral applications.
• the drug transporter inhibitor may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for capsules, tablets, pellets, suppositories, and any other form suitable for use.
• the carriers which can be used are water, glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium, trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea and other carriers suitable for use in manufacturing preparations, in solid or semisolid form, and in addition auxiliary, stabilizing, thickening and coloring agents and perfumes may be used.
• the drug transporter inhibitor alone or in conjunction with a therapeutic agent, is included in the pharmaceutical composition or dosage form in an amount sufficient to produce the desired effect upon the process or condition, including a variety of conditions and diseases in humans.
• the drug transporter inhibitor for preparing solid compositions such as tablets, is mixed with a pharmaceutical carrier, e.g., conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g., water, to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention, or a non-toxic pharmaceutically acceptable salt thereof.
• a pharmaceutical carrier e.g., conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g., water, to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention, or a non-toxic pharmaceutically acceptable salt thereof
• the drug transporter inhibitor alone or in conjunction with therapeutic agent, is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as capsules, tablets, caplets, or pills.
• the capsules, tablets, caplets, or pills of the novel pharmaceutical composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action.
• the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former.
• the two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release.
• enteric layers or coatings such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.
• Controlled release (e.g., slow- release or sustained-release) dosage forms, as well as immediate release dosage forms are specifically contemplated according to the present invention.
• compositions in liquid forms in which a therapeutic agent may be incorporated for administration orally or by injection include aqueous solution, suitable flavored syrups, aqueous or oil suspensions, and emulsions with acceptable oils such as cottonseed oil, sesame oil, coconut oil or peanut oil, or with a solubilizing or emulsifying agent suitable for intravenous use, as well as elixirs and similar pharmaceutical vehicles.
• suitable dispersing or suspending agents for aqueous suspensions include synthetic and natural gums such as tragacanth, acacia, alginate, dextran, sodium carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone or gelatin.
• compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders.
• the liquid or solid compositions may contain suitable pharmaceutical lv acceptable excipients as set out above.
• the compositions are administered by the oral or nasal respiratory route for local or systemic effect.
• Compositions in preferably sterile pharmaceutically acceptable solvents may be nebulized by use of inert gases. Nebulized solutions may be breathed directly from the nebulizing device or the nebulizing device may be attached to a face mask, tent or intermittent positive pressure breathing machine.
• Solution, suspension or powder compositions may be administered, preferably orally or nasally, from devices which deliver the formulation in an appropriate manner.
• a drug transporter inhibitor alone, or in combination with a therapeutic agent may be administered to the human subject by known procedures including but not limited to oral, sublingual, intramuscular, subcutaneous, intravenous, intratracheal, transmucosal, or transdermal modes of administration. When a combination of these compounds are administered, they may be administered together in the same composition, or may be administered in separate compositions. If the therapeutic agent and the drug transporter inhibitor are administered in separate compositions, they may be administered by similar or different modes of administration, or may be administered simultaneously with one another, or shortly before or after the other.
• the drug transporter inhibitors alone, or in combination with therapeutic agents are formulated in compositions with a pharmaceutically acceptable carrier ("pharmaceutical compositions").
• the carrier must be "acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
• suitable pharmaceutical carriers include lactose, sucrose, starch, talc, magnesium stearate, crystalline cellulose, methyl cellulose, carboxymethyl cellulose, glycerin, sodium alginate, gum arabic, powders, saline, water, among others.
• the formulations may conveniently be presented in unit dosage and may be prepared by methods well-known in the pharmaceutical art, by bringing the active compound into association with a carrier or diluent, or optionally with one or more accessory ingredients, e.g., buffers, flavoring agents, surface active agents, or the like.
• the choice of carrier will depend upon the route of administration.
• compositions may be administered as solid or semisolid formulations, including as capsules, tablets, caplets, pills or patches.
• Formulations may be presented as an immediate-release or as a controlled-release (e.g., slow-release or sustained-release) formulation, including, for example, methadone hydrochloride, Dolophine (Roxane); Methadose (Mallinkrodt); hydrocodone bitartrate and acetaminophen (Nicodin, Knoll Labs); Lortab (UCB); oxycodone hydrochloride, OxyContin, sustained release (Purdue); tramadol (Ultram, Johnson & Johnson); meperidine hydrochloride (Demerol, Sanofi); hydromorphone hydrochloride (Dilaudid, Knoll Labs); codeine sulfate (Roxane); or propoxyphene hydrochloride (Darvon, Lilly).
• methadone hydrochloride Dolophine (Roxane); Methadose (
• the formulation may be presented as capsules, tablets, caplets, powders, granules or a suspension, with conventional additives such as lactose, mannitol, corn starch or potato starch; with binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch, gelatins, natural sugars such as glucose or beta- lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, or the like; with disintegrators such as corn starch, potato starch, methyl cellulose, agar, bentonite, xanthan gums, sodium carboxymethyl-cellulose or the like; or with lubricants such as talc, sodium oleate,. sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride or the like.
• conventional additives such as lactose, mannitol, corn
• the compounds may be combined with skin penetration enhancers such as propylene glycol, polyethylene glycol, isopropanol, ethanol, oleic acid, ⁇ -methylpyrrolidone, or the like, which increase the permeability of the skin to the compounds, and permit the compounds to penetrate through the skin and into the bloodstream.
• skin penetration enhancers such as propylene glycol, polyethylene glycol, isopropanol, ethanol, oleic acid, ⁇ -methylpyrrolidone, or the like, which increase the permeability of the skin to the compounds, and permit the compounds to penetrate through the skin and into the bloodstream.
• the compound/enhancer compositions also may be combined additionally with a polymeric substance such as ethylcellulose, hydroxypropyl cellulose, ethylene/ vinylacetate, polyvinyl pyrrolidone, or the like, to provide the composition in gel form, which can be dissolved in solvent such as methylene chloride, evaporated to the desired viscosity, and
• the compounds may combined with a sterile aqueous solution which is preferably isotonic with the blood of the recipient.
• a sterile aqueous solution which is preferably isotonic with the blood of the recipient.
• Such formulations may be prepared by dissolving solid active ingredient in water containing physiologically compatible substances such as sodium chloride, glycine, or the like, and/or having a buffered pH compatible with physiological conditions to produce an aqueous solution, and/or rendering said solution sterile.
• the formulations may be present in unit or multi-dose containers such as sealed ampoules or vials.
• the amount of the therapeutic agent administered may be a therapeutic or sub- therapeutic amount.
• a "therapeutic" amount is the amount of the therapeutic agent which causes a therapeutic effect in a subject administered the therapeutic agent alone.
• the amount of the drug transporter inhibitor may be an amount effective to enhance the therapeutic potency of and/or attenuate the adverse side effects of the therapeutic agent.
• the optimum amounts of the drug transporter inhibitor administered alone or in combination with a therapeutic agent will of course depend upon the particular drug transporter inhibitor and therapeutic agent used, the carrier chosen, the route of administration, and/or the pharmacokinetic properties of the subject being treated.
• the amount of the drug transporter inhibitor administered is an amount effective to enhance or maintain the therapeutic potency of the therapeutic agent and/or attenuate or maintain the adverse side effects of the therapeutic agent. This amount is readily determinable by one skilled in the art according to the invention.
• Compounds can be tested in vitro for their ability to serve as inhibitors of drug transporter proteins.
• Cells expressing a drug transporter such as P-glycoprotein, are suitable for use in in vitro screens. Details of an appropriate protocol for testing compounds for their ability to inhibit PGP-associated drug transport are given in the Examples.
• the method described involves growing a monolayer of PGP-expressing cells in such a manner as to present PGP on only one face of the monolayer, then applying a known PGP substrate and the test substance to the PGP-presenting side of the monolayer. After a period of incubation, the level of PGP substrate is measured on the non-PGP-presenting side of the monolayer.
• Inhibition of the drug transporter protein is characterized by a decreased concentration of PGP substrate on the non-PGP-presenting side of the monolayer as compared to the concentration found if the experiment is performed in the absence of test substrate.
• inhibitors of drug transporter proteins can be identified by assaying for ATPase activity. In this type of assay, the ability of the test substrate to inhibit the ATPase activity of a drug transporter activated by a known substrate is examined. The test substances are incubated with ABC drug transporter containing-membranes and supplemented with MgATP, with and without sodium orthovanadate present. Orthovanadate inhibits PGP by trapping MgADP in the nucleotide binding site. Thus, the ATPase activity measured in the presence of orthovanadate represents non-PGP ATPase activity and was subtracted from the activity generated without orthovanadate to yield vanadate-sensitive
• Porcine kidney-derived, LLC-PK l5 cells expressing human PGP cDNA were cultured in 24 well TranswellTM culture inserts at 37° C on an orbital shaker. Transport assays were conducted in 24 well TranswellTM culture inserts with Hanks Balanced Salt Solution (HBSS) buffered with the addition of 10 mM HEPES (pH 7.2).
• HBSS Hanks Balanced Salt Solution
• 10 mM HEPES pH 7.2
• a sample from the receiver chamber was analyzed for the amount of digoxin present.
• the positive control for inhibition was 25 ⁇ M ketoconazole added to donor and receiver chambers with 5 ⁇ M [ 3 H]- digoxin added to the donor chamber.
• the negative control for inhibition was 5 ⁇ M [ 3 H]- digoxin added to the donor chamber (either the apical or basolateral chamber depending on the direction of transport) with Hanks Balanced Salt Solution (HBSS) buffered with the addition of 10 mM HEPES (pH 7.2) and DMSO at 0.55% in the receiver chamber.
• HBSS Hanks Balanced Salt Solution
• the rate of digoxin transported from the apical chamber to the basolateral chamber (A to B) and from the basolateral chamber to the apical chamber (B to A) was measured and apparent permeability P app constants calculated.
• the polarization ratio P app B t o A Papp A was calculated.
• a lower polarization ratio in the 15B-J cells with test substance relative to that without test substance provides evidence for inhibition of PGP-mediated digoxin transport by the test substance.
• Transport of 5 ⁇ M [3H]-digoxin was measured following coincubation with the test substances at nominal concentrations in the range of 0 to 100 ⁇ M.
• Naloxone and naltrexone exhibited inhibitory behavior at the 30 and 100 ⁇ M concentrations.
• Digoxin transport appears to have been slightly inhibited at naloxone and naltrexone concentrations below 30 ⁇ M, however the inhibition was not concentration- dependent.
• Digoxin transport was increasingly inhibited in response to increasing concentration of nalmefene at concentrations between 3 and 100 ⁇ M.
• the positive control 25 ⁇ M ketoconazole, inhibited digoxin transport within the accepted range, indicating that the cell model performed as expected.
• Example 2 6- ⁇ -Naltrexol Does Not Inhibit Human PGP-Mediated Transport
• Porcine kidney-derived, LLC-PK l5 cells expressing human PGP cDNA were cultured in 24 well TranswellTM culture inserts at 37° C on an orbital shaker.
• Transport assays were conducted in 24 well TranswellTM culture inserts with Hanks Balanced Salt Solution (HBSS) buffered with the addition of 10 mM HEPES (pH 7.2).
• HBSS Hanks Balanced Salt Solution
• test substance 6- ⁇ -naltrexol
• LC Resources, Inc Stock solutions of the compounds were made in DMSO, and dilutions of these in transport buffer were prepared for assay in the monolayers.
• the DMSO concentration (0.55%) was constant for all conditions within the experiment.
• test substance was added to the donor and receiver chambers. Duplicate monolayers and thirteen test substance concentrations of 0.0001, 0.0003, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30 and 100 ⁇ M, were used.
• PGP substrate [ 3 H]-digoxin, at 5 ⁇ M was added to the donor chamber (either the apical or basolateral chamber depending on the direction of transport). After an incubation time of 90 minutes, a sample from the receiver chamber was analyzed for the amount of digoxin present. The positive control for inhibition was 25 ⁇ M ketoconazole added to donor and receiver chambers with 5 ⁇ M [ 3 H]- digoxin added to the donor chamber.
• the negative control for inhibition was 5 ⁇ M [ 3 H]- digoxin added to the donor chamber (either the apical or basolateral chamber depending on the direction of transport) and Hanks Balanced Salt Solution (HBSS) buffered with the addition of 10 mM HEPES (pH 7.2) and DMSO at 0.55% in the receiver chamber.
• HBSS Hanks Balanced Salt Solution
• test substance 6- ⁇ -naltrexol was not a potent inhibitor of PGP-mediated digoxin transport, in the concentration range tested.
• Example 3 - Opioid Receptor Antagonists inhibit PGP ATPase Activity
• test substances naloxone, naltrexone and nalmefene
• DMSO fetal sulfate
• transport buffer fetal sulfate
• DMSO concentration 0.55% was constant for all conditions within the experiment. All test substance and control drug solutions prepared in HBSS/HEPES buffer contained 0.55% DMSO.
• test substances were incubated in the membranes and supplemented with MgATP, with and without sodium orthovanadate present.
• Orthovanadate inhibits PGP by trapping MgADP in the nucleotide binding site.
• the ATPase activity measured in the presence of orthovanadate represents non-PGP ATPase activity and was subtracted from the activity generated without orthovanadate to yield vanadate-sensitive ATPase activity.
• ATPase assays were conducted in 96-well microtiter plates.
• a 0.06 ml reaction mixture containing 40 ⁇ g PGP membranes, test substance, and 4 mM MgATP, in buffer containing 50 mM Tris-MES, 2 mM EGTA, 50 mM KCl, 2 mM dithiothreitol, and 5 mM sodium azide, plus organic solvent was incubated at 37°C for 20 minutes.
• Triplicate incubations of ten test substance concentrations (of 0.003, 0.01, 0.03, 0J, 0.3, 1.0, 3.0, 10, 30 and 100 ⁇ M) and the test vehicle without drug, were used.
• the liberation of inorganic phosphate was detected by its absorbance at 800 nm and quantitated by comparing the absorbance to a phosphate standard curve.
• concentration dependence of the PGP was analyzed for evidence of saturation of PGP- ATPase activity, and apparent kinetic parameters were calculated by non-linear regression.
• the positive control for stimulation of ATPase activity was 20 ⁇ M verapamil, and the positive control for inhibition of basal ATPase activity was 25 mM ketoconazole.
• the order of inhibition of the PgP 1 a associated ATPase was nalmefene, naltrexone and naloxone. Naloxone only weakly inhibited the PGP la associated ATPase. None of the compounds were stimulators of ATPase.
• a molecular modeling analysis was performed on a series of compounds, including opioid analogues, to elucidate their mode of interaction with PGP- la, and to determine, if possible, a pharmacophore for drug transporter inhibitors useful according to the present invention.
• Exemplary compounds in this study were naltrexone, naloxone, nalmefene, 6- ⁇ - naltrexol and nalorphine.
• the structures of compounds are illustrated in Fig. 1. The compounds are structurally very similar, and exhibit two measured activities.
• “Activity 1" is characterized by a low capacity, high affinity binding site with activity ranging from 0.3 nM to greater than 200 ⁇ M.
• activity 2 is characterized by a high capacity, low affinity binding site with activity ranging from 10 ⁇ M to greater than 100 ⁇ M.
• Table 6 provides the biological activities for each of the exemplary compounds.
• Table 6 Biological Activity of Exemplary Compounds
• nalorphine exhibits no measurable activity.
• the structures of the compounds as represented in the Merck Index represent the active form of the compound.
• An important difference in these compounds is that nalo ⁇ hine lacks the hydroxyl group in the central ring at position 14 (see, e.g., Figure 1), indicating that this hydroxyl group is a requirement for activity.
• the most active compounds nalmefene and naltrexone
• This moiety may be viewed as a necessary, but not sufficient condition, since several of the inactive compounds also possess this hydrophobic region.
• 6- ⁇ -Naltrexol is even less active is attributed to the hydroxyl substituent at the 6 position being oriented ⁇ to the ring system, perhaps penetrating a sterically limited region in the receptor.
• the analysis indicates that the presence of the hydroxyl group at the 14- position may be required for activity, since nalo ⁇ hine, with no measured activity, lacks this moiety.
• the two most active compounds possess an ethylene group and a carbonyl group respectively at the 6-position.
• This may represent a requirement for electron density at this position, rather than a hydrogen-bond acceptor site, as there is only a one order of magnitude difference in activity (0.3nM vs. 3nM) between the ethylene group (nalmefene) and the carbonyl group (naltrexone).
• 6- ⁇ -Naltrexol places its hydroxyl group in a direction that penetrates into this region.
• a hydrophobic group is required as the N-substituent for highest activity, as naloxone, with a double bond rather than the cyclopropyl group, exhibits significantly lower activity.
• the intestinal permeability coefficients of the Kamm compounds were studied using Caco-2 monolayers and reverse-phase HPLC method for quantitation. Further the efflux ratios (transport from B to A:transport from A to B) were calculated. The efflux ratios for a selection of the Kamm compounds measured at 250 ⁇ M are provided in Table 8.
• Fig. 2 An overlay of the opioid analogue structures is presented in Fig. 2. All active (“Activity 1") compounds share the following features: two hydroxyl groups (a) at positions 3 and 14, a furan ring system, a hydrophobic region in ring system, a region of electron density at position 6 (b), and a cyclic tertiary nitrogen (c) with an appended hydrophobic group (d).
• the Kamm et al data was combined with the high affinity/low capacity data provided for the exemplary opioid compounds.
• the 200 "nearest neighbors" are listed in Table 11 below. Note that in the Receptor-Relevant Subspace, the active compounds are focused in a small region of the overall chemistry space.
• a pharmacophore for a drug transporter inhibitor useful according to the present invention contains the hydroxyl groups at the 14-position and 3 -position as discussed above, the nitrogen, the hydrophobic region (tethered to the nitrogen), and the region of electron density at the 6-position. Other combinations of features are also possible as discussed below.
• the distance between the hydroxuyl groups in the pharmacophore ("H" of OH to "H” of OH) is approximately 7.4 A.
• the equivalent distance in "Kamm 1" is ⁇ 7.7 A. These distances are to the Hydrogen atoms, rather than the H-bond acceptors in the binding site.
• the N-substituent lengths of nalmefene (from N to terminal Carbons) are ⁇ 3.9 A and -3.5 A.
• N-substituent length of naloxone (from N to terminal Carbon) is ⁇ 3.4 A.
• the three-dimensional coordinates of naltrexone are provided in Table 12. Table 12: Three-dimensional coordinates
• a pharmacophore may be defined by: (1) a hydrogen bonding moiety at a three-dimensional location corresponding to the hydroxyl at position 3 of naltrexone; (2) a hydrogen bonding moiety at a three-dimensional location corresponding to the hydroxyl at position 14 of naltrexone; (3) a hydrophobic moiety at a three-dimensional location corresponding to the cyclopropyl moiety appended to the nitrogen of naltrexone; and (4) a region of electron density at a three-dimensional location corresponding to the ethylene moiety at 6-position of naltrexone.
• a heparinized saline solution (100 IU/ml) was maintained in the arterial cannulae to prevent clotting. All ends of the catheters were passed subcutaneously to a plastic cup placed on the surface of the neck out of reach from the rat. Two stainless steel sutures are placed in the tail of the rat 1 and 3 cm from the root of the tail for the analgesic measurements.
• the rat is placed in a CMA/120 system for freely moving animals with free access to water and food, and the experiment started approximately 24 hours later. All experiments started at the same time of the day.
• Each rat was weighed (range 270 - 330g).
• the baseline for antinociception was measured three times with 15-minute intervals before the start of the experiment. During the procedure all rats were held gently in a towel.
• the duration of the stimuli was 1 sec, using a frequency of electrical square waves of 125 pulses/sec and a pulse width of 1.6 msec.
• the voltage was increased in logarithmic steps. A vocalization response was recorded as the endpoint, the pain threshold.
• the maximal voltage accepted was 11.5 N.
• Mo ⁇ hine was administered as an intravenous infusion over 10 minutes at an infusion rate of 1.8 mg/kg/h (0J mg/kg) and 18 mg/kg/h (1 mg/kg).
• 200 ⁇ l blood samples were collected at 5, 10, 15, 20, 40, 60, 120, 240 and 300 min from the start of the 10-minute infusion. All blood samples were centrifuged at 5,000 rpm for 5 minutes, and the plasma harvested and stored at -20°C until analyzed.
• Antinociception was measured 5, 10, 15, 20, 30, 45, 60 min after the start of the infusion and thereafter every 30 minutes up to 240 min.
• the ratio of brain concentration of mo ⁇ hine to blood concentration of mo ⁇ hine is shown the Fig. 5.
• the animals are anaesthetized by inhalation of Enfluran®.
• a heparinised saline solution (100 IU/ml) was maintained in the arterial cannulae to prevent clotting.
• the blood probe (CMA/20) was inserted into the right jugular vein through a guide cannulae and fixed to the pectoralis muscle with two sutures.
• the rat was placed in a stereotaxic instrument for the implantation of the striatal probe.
• the brain probe was inserted into the hole and fixed with a screw and dental cement.
• a 15 cm PE-50 tubing was looped subcutaneously on the back of the rat to the surface of the neck in order to let the perfusion solution adjust to body temperature before entering the brain probe. All ends of the catheters were passed subcutaneously to a plastic cup placed on the surface of the neck out of reach from the rat.
• Each rat was weighed (range 270 - 330g).
• the probes were perfused with blank Ringer's solution at a flow rate of 1 ⁇ l/min. Microdialysate fractions were collected every 15 minutes for 1 hour. After a 60 min stabilization period the microdialysis probes were perfused with an mo ⁇ hine solution containing 100 ng/ml (blood) or 200 ng/ml (striatum).
• Unbound concentrations of mo ⁇ hine were calculated from the dialysate concentration of mo ⁇ hine adjusted for the in vivo recovery value. After the retrodialysis period, the probes were perfused for one hour with blank perfusion solution.
• the rats were randomly assigned into two groups receiving an i.v. infusion of mo ⁇ hine hydrochloride over 10 min.
• Microdialysates were collected over a period of 240 min into pre- weighed vials. Samples were taken at 5 min intervals during the infusion, at 10 min intervals over the following hour and at 15 min intervals over the remaining 3 hours. Dialysates were collected, weighed and stored at -20°C until analyzed. Arterial blood samples (200 ul) were collected in heparinised vials at 0, 8, 20, 70, 130, 190 and 240 min. After collection, samples were centrifuged at 5000 rpm for 5 min, and plasma was harvested and frozen at - 20°C until analysis.
• Antinociception was measured according to the electrical stimulation vocalization method.
• An electrical stimulus was applied to the two electrodes implanted in the tail of the rat. During the procedure all rats were held gently in a towel. The duration of the stimuli was 1 sec, using a frequency of electrical square waves of 125 pulses/sec and a pulse width of 1.6 msec. The voltage was increased in logarithmic steps. A vocalization response was recorded as the endpoint, the pain threshold. The maximal voltage accepted was 11.5 N. The baseline value of the pain threshold was estimated three times at 15 min intervals before the start of the experiment. Antinociception was recorded at the end of the blank, retrodialysis and washout periods and at 5, 10, 15, 20, 30, 45, 60 min after the start of the infusion and thereafter every 30 minutes up to 240 min.
• the blood gas status of the rats was monitored by injection of a 50 ⁇ l arterial blood sample into a blood gas analyzer to determine the arterial pO 2 , pCO 2 , O 2 saturation and pH. During the experiment the blood gas status was monitored just before antinociception measurement.
• each rat was weighed (range 270 - 330g).
• the probes were perfused with blank Ringer's solution at a flow rate of 1 ⁇ l/min. Microdialysate fractions were collected every 15 minutes for 1 hour. After a 60 min stabilization period the microdialysis probes were perfused with an mo ⁇ hine solution containing 100 ng/ml (blood) or 200 ng/ml
• mice received an i.v. infusion over 10 minutes of mo ⁇ hine hydrochloride or mo ⁇ hine and naltrexone as determined above. After the infusion was stopped at Day 4, the remaining three rats of groups M2 and F2 were decapitated, directly after directly after mo ⁇ hine and ⁇ TX administration. The brain was divided into two parts and each part put in a plastic cup and stored at -70°C until analyzed.
• Example 6 - Tolerance and Withdrawal in Mice 40 male mice were randomized into 5 groups of eight. All mice were administered single 3mg/kg mo ⁇ hine daily (b.i.d.), beginning on Day 1.
• the anti-nociceptive effect was assayed by standard tail flick procedures for mice in Group 1 on Days 1, 3, 5, 8, 10 and 12.
• the mice in Groups 2-5 were assayed on Days 5, 8, 10 and 12.
• Groups 2, 3, 4 and 5 received daily doses of 3 ng/kg, 30 ng/kg, 300 ng/kg and 3000 ng/kg naltrexone (b.i.d.), respectively, beginning at day 6.
• Anti-nociceptive effect was assayed by tail flick on Days 5 (prior to naltrexone dosing), 6, 8, and 10.
• the mice in Group 1 showed adaptation to the repeated dosing of mo ⁇ hine.
• the data were subjected to two types of analyses; cross sectional time series analysis using generalized estimating equations (GEE) and survival analysis using Cox regression.
• GEE generalized estimating equations
• the Cox and GEE analyses of Group l were consistent and showed that the latency (time to tail flick) was shorter after day 1.
• the Cox analysis showed that although Day was the major factor influencing latency, Time periods after 60 minutes also significantly influenced latency. Note it could be argued that reduced latency after day 1 is an adaptation to the mo ⁇ hine or an adaptation by the mice to having repeated tail flick experiments conducted on them.
• mice had similar tail flick responses on day 5 and this is despite the fact that four groups (groups 2-5) experienced the tail flick experiment for first time, this can be inte ⁇ reted as evidence that reduced latency after day 1 is due to adaptation to mo ⁇ hine.
• mice 40 female mice were randomized into 5 groups of eight. All mice were administered single 3mg/kg mo ⁇ hine daily (b.i.d.), beginning on Day 1.
• the anti-nociceptive effect was assayed by standard tail flick procedures for mice in Group 1 on Days 1, 3, 5, 8, 10 and 12.
• the mice in Groups 2-5 were assayed on Days 5, 8, 10 and 12.
• Groups 2, 3, 4 and 5 received daily doses of 3 ng/kg, 30 ng/kg, 300 ng/kg and 3000 ng/kg naltrexone (b.i.d.), respectively, beginning at day 6.
• Anti-nociceptive effect was assayed by tail flick on Days 5 (prior to naltrexone dosing), 6, 8, and 10.
• mice in group 1 The adaptation of mice in group 1 to the repeated daily dosing with mo ⁇ hine was analyzed. As with male mice, female mice show an adaptation to mo ⁇ hine especially from day five onwards. The change in latency within a day (dayl) of group 1 mice was analyzed. At all times after time zero latency was significantly longer than for time zero. The variability between groups of mice were compared at Day 5. There were little differences between groups of female mice on day 5.
• naltrexone effect at Days 6, 8, and 10 was analyzed. All concentrations (above zero) of naltrexone were significantly different from zero in enhancing latency. In females, 30 ng appears to be significantly better than other concentrations at enhancing latency. Group C
• mice 40 male mice were randomized into 5 groups of eight. All mice were administered single 3mg/kg mo ⁇ hine daily (b.i.d.), beginning on Day 1. In addition, Groups 2, 3, 4 and 5 received daily doses of 3 ng/kg, 30 ng/kg, 300 ng/kg and 3000 ng/kg naltrexone (b.i.d.), respectively, beginning at day 1. The anti-nociceptive effect was assayed by standard tail flick procedures for all mice in on Days 1, 3, 5, 8, and
• mice 40 female mice were randomized into 5 groups of eight. All mice were administered single 3mg/kg mo ⁇ hine daily (b.i.d.), beginning on Day 1. In addition Groups 2, 3, 4 and 5 received daily doses of 3 ng/kg, 30 ng/kg, 300 ng/kg and 3000 ng kg naltrexone (b.i.d.), respectively, beginning at day 1. The anti-nociceptive effect was assayed by standard tail flick procedures for all mice in on Days 1, 3, 5, 7, and 10. On Day 11, every mouse received a single bolus dose of 10 ⁇ g/kg naltrexone in addition to the existing mo ⁇ hine/naltrexone regimen.
• naltrexone The response to small doses of naltrexone was measured. Although a dose 0.3 ng/kg of naltrexone gave the longest latency, this was not significantly different from 0.03 or to 3 ng/kg of naltrexone (Table 13).
• naltrexone at concentrations of 30 ng/kg and 300 ng/kg gave significantly longer latency than other concentrations of naltrexone (FIG. 6 and Table 15).
• naltrexone administered to male mice Groups A and C, on days 8 and 10
• naltrexone at 300 ng gave the greatest latency and was significantly different to other concentrations of naltrexone.
• mice in Group 2 3 mg/kg of mo ⁇ hine was administered as a single bolus dose to each mouse on group 1 on a daily basis. Mice in Group 2 were also administered 3 mg/kg mo ⁇ hine daily. In addition to the mo ⁇ hine, mice in Group 2 also received 3 ng/kg naltrexone daily beginning on Day 6. At Day 15, the dose of naltrexone was lowered to 0J ng/kg.
• mice showed distinct tolerance to the mo ⁇ hine.
• administration of the naltrexone broke the tolerance (FIG. 7).
• mice were administered 0.1 mg/kg oxycodone plus either 1 ng/kg naltrexone, 1 ng/kg nalmefene or 1 pg/kg of nor-BNI. In all cases the mice did not develop tolerance to the oxycodone.
• female mice were administered either 1 mg/kg or 5 mg/kg oxycodone in combination with 1 pg/kg, 1 ng/kg or 1 ⁇ g/kg naltrexone or 1 pg/kg nor-BNI. None of the mice developed tolerance to the oxycodone. Additionally, the male and female mice who had developed a tolerance to oxycodone were administered a single 10 ⁇ g/kg dose of naloxone.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Veterinary Medicine (AREA)
• Public Health (AREA)
• General Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Animal Behavior & Ethology (AREA)
• Medicinal Chemistry (AREA)
• Pharmacology & Pharmacy (AREA)
• Epidemiology (AREA)
• Engineering & Computer Science (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Organic Chemistry (AREA)
• General Chemical & Material Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Pain & Pain Management (AREA)
• Neurosurgery (AREA)
• Neurology (AREA)
• Biomedical Technology (AREA)
• Psychiatry (AREA)
• Biophysics (AREA)
• Molecular Biology (AREA)
• Emergency Medicine (AREA)
• Rheumatology (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Applications Claiming Priority (7)

Publications (1)

Family

ID=27399762

Family Applications (1)

Country Status (5)

Families Citing this family (2)

Citations (12)

• 2001
  • 2001-10-30 CA CA002427330A patent/CA2427330A1/en not_active Abandoned
  • 2001-10-30 AU AU2002239427A patent/AU2002239427A1/en not_active Abandoned
  • 2001-10-30 WO PCT/US2001/045367 patent/WO2002041884A2/en active Search and Examination
  • 2001-10-30 JP JP2002544063A patent/JP2004528273A/ja active Pending
  • 2001-10-30 EP EP01987187A patent/EP1392265A2/en not_active Withdrawn

Patent Citations (13)

Non-Patent Citations (7)

Also Published As

Similar Documents

Legal Events