Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
• • 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
• • 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
  • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
  • A61K31/5375—1,4-Oxazines, e.g. morpholine
  • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • 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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D217/00—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems
  • C07D217/12—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems with radicals, substituted by hetero atoms, attached to carbon atoms of the nitrogen-containing ring
  • C07D217/14—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems with radicals, substituted by hetero atoms, attached to carbon atoms of the nitrogen-containing ring other than aralkyl radicals
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
  • C07D401/10—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing aromatic rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
  • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
  • C07D403/10—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing aromatic rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
  • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
  • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
  • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
  • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value
  • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/20—Dermatological disorders

Definitions

• the present invention relates to methods of using histamine 3 receptor (H3R) antagonists to treat pruritus or itch in an individual and methods for using H3R to screen for compounds capable of treating pruritus in an individual and methods for using pruritus/itch model systems for screening H3R antagonists for their usefulness in treating pruritus.
• H3R histamine 3 receptor
• Pruritus or itch is the unpleasant sensation that leads to a desire to scratch (for reviews, see Journal of Investigative Dermatology (2006) 126: 1705-1718; and Lancet (2003) 361 : 690-94). It is a common and distressing symptom in a variety of conditions and diseases. Pruritus typically occurs in peripheral diseases, such as, allergic conjunctivitis, allergic rhinitis, hemorrhoids, atopic dermatitis, allergic dermatitis, acute and chronic urticaria (hives), psoriasis and dermatoses of fungal, allergic and non-allergic origin.
• peripheral diseases such as, allergic conjunctivitis, allergic rhinitis, hemorrhoids, atopic dermatitis, allergic dermatitis, acute and chronic urticaria (hives), psoriasis and dermatoses of fungal, allergic and non-allergic origin.
• Itching can also be a major symptom of many systemic diseases such as, Hodgkin's disease, chronic renal failure, polycythemia vera, hyperthyroidism, malignancy, infection, chronic cholestatic liver disease, and end-stage renal disease, and cholestasis.
• systemic diseases such as, Hodgkin's disease, chronic renal failure, polycythemia vera, hyperthyroidism, malignancy, infection, chronic cholestatic liver disease, and end-stage renal disease, and cholestasis.
• senile itch without an obvious cause, except perhaps xerosis, occurs in more than half of the population aged 70 years. In all cases chronic severe generalized itch can be disabling.
• PBC primary biliary cirrhosis
• PSC primary sclerosis cholangitis
• Chronic renal disease epidural morphine
• pregnancy diabetes mellitus, thyroid illness, hyperparathyroidism, iron deficiency anemia, viral infection, aquagenic pruritus, and psychogenic pruritus.
• Pruritus causes sufferers to scratch, leading to skin damage, increased risk of skin infection, and worsening of inflammation.
• the pathogenesis of pruritus is not well understood, and treatment options are limited (Paus, R., et al., J. Clin. Invest. 2006, 116: 1174-1185).
• pruritus was once thought to be a subliminal form of pain (intensity theory), current evidence points to separate sensory neuronal systems mediating the two modalities.
• pain and pruritus are dissociable. Pain and pruritus evoke different motor responses, scratching for pruritus and withdrawal for pain.
• systemically-administered opioids have a dichotomous effect on these two sensory modalities.
• ⁇ -Opioid receptor agonists reduce pain but can cause pruritus.
• antagonizing the central mu-opioid receptors for example with naloxone or naltrexone, suppresses pruritus and at the same time may lower the pain threshold.
• Pruritus due to skin inflammation is thought to be mediated at least partly by activation of skin mast cells, which release pruritogenic mediators to activate receptors on peripheral nerve endings to transmit itch signals.
• histamine is a particularly potent pruritogen. Histamine injected into the skin causes strong itch sensations in humans and animals. Therefore, antagonists of the histamine receptors have been explored as pruritus treatments.
• one aspect of the present invention relates to the inhibition of the H3R in an individual, such as by administration of a compound or agent of the present invention, can reduce itching or pruritus.
• the G-protein coupled histamine 3 receptor H3R
• the present invention provides a method of preventing and/or treating pruritus in an individual in need thereof by administering a therapeutically effective amount of a compound or agent that modulates the H3R.
• peripherally restricted antagonists of H3R are capable of mediating the inhibition of itch. Accordingly, screening for peripherally restricted antagonists and the application of peripherally restricted antagonists in the various embodiments of the invention is contemplated. Peripherally restricted compounds may be advantageous to the extent that the peripheral restriction reduces the CNS effects of H3R inhibition. Such effects may include, for example, wakefulness.
• H3R antagonists are peripherally restricted and may be assayed or screened based on their inability or reduced ability to inhibit H3R in the CNS (for example, in the brain).
• the invention comprises a method for treating or preventing itching or the symptoms thereof in an individual wherein an antagonist of H3R is administered to the individual.
• the invention disclosed herein is suitable for the prevention and/or treatment of pruritus that is associated with a disease or disorder, such as those described herein.
• One aspect of the present invention encompasses every combination of one or more diseases or disorders for which itch is associated selected from the group: eczema, atopic eczematous dermatitis, seborrheic dermatitis, atopic dermatitis, contact dermatitis, irritant dermatitis, xerosis (dry skin), psoriasis, a fungal infection, athlete's foot, a yeast infection, diaper rash, vaginal itch, parasitic infections, parasitic infestations including scabies and lice, lichen planus, lichen simplex, lichen simplex chronicus, lichen sclerosis, itch secondary to medications, senile itch, uremia, idiopathic itch, itch associated with liver cirrhosis, itch associated with inflammation, itch associated with allergies, itch associated with cancer, itch associated with chemotherapy, itch associated with kidney disease, itch associated with haemodialysis, burns, scalds, sunburn, wound
• GPCR G protein-coupled receptor
• GPCRs represent an important area for the development of pharmaceutical products. Drugs active at GPCRs have therapeutic benefit across a broad spectrum of human diseases as diverse as pain, cognitive dysfunction, hypertension, peptic ulcers, rhinitis, and asthma. Of the approximately 500 clinically marketed drugs, greater than 30% are modulators of GPCR function. These drugs exert their activity at approximately 30 well-characterized GPCRs. (See, e.g., Wise et al, Annu Rev Pharmacol Toxicol (2004) 44:43-66.)
• GPCRs share a common structural motif, having seven sequences of between 22 to 24 hydrophobic amino acids that form seven alpha helices, each of which spans the membrane (each span is identified by number, i.e., transmembrane- 1 (TM-1), transmembrane-2 (TM-2), etc.).
• the transmembrane helices are joined by strands of amino acids between transmembrane-2 and transmembrane- 3, transmembrane-4 and transmembrane-5, and transmembrane-6 and transmembrane-7 on the exterior, or "extracellular" side, of the cell membrane (these are referred to as "extracellular" regions 1, 2 and 3 (EC-1, EC-2 and EC-3), respectively).
• transmembrane helices are also joined by strands of amino acids between transmembrane- 1 and transmembrane-2, transmembrane- 3 and transmembrane-4, and transmembrane-5 and transmembrane-6 on the interior, or "intracellular” side, of the cell membrane (these are referred to as "intracellular” regions 1, 2 and 3 (IC-1, IC-2 and IC-3), respectively).
• the "carboxy" (“C”) terminus of the receptor lies in the intracellular space within the cell, and the "amino" (“N”) terminus of the receptor lies in the extracellular space outside of the cell.
• GPCR "promiscuous" with respect to G proteins, i.e., that a GPCR can interact with more than one G protein. See, Kenakin, Life Sciences (1988) 43: 1095-1101. Although other G proteins exist, currently, Gq, Gs, Gi, Gz, and Go are G proteins that have been identified. Ligand-activated GPCR coupling with the G-protein initiates a signaling cascade process (referred to as “signal transduction"). Under normal conditions, signal transduction ultimately results in cellular activation or cellular inhibition. Current understanding is that the IC-3 loop as well as the carboxy terminus of the receptor interacts with the G protein.
• G proteins which appear to couple several classes of GPCRs to the phospholipase C pathway, such as G15 or G16 (Offermanns & Simon, J Biol Chem (1995) 270: 15175-80), or chimeric G proteins designed to couple a large number of different GPCRs to the same pathway, e.g. phospholipase C (Milligan & Rees, Trends in Pharmaceutical Sciences (1999) 20: 118-24).
• G15 or G16 Offermanns & Simon, J Biol Chem (1995) 270: 15175-80
• chimeric G proteins designed to couple a large number of different GPCRs to the same pathway, e.g. phospholipase C (Milligan & Rees, Trends in Pharmaceutical Sciences (1999) 20: 118-24).
• GPCRs exist in the cell membrane in equilibrium between two different conformations: an "inactive" state and an “active” state.
• a receptor in an inactive state is unable to link to the intracellular signaling transduction pathway to initiate signal transduction leading to a biological response.
• Changing the receptor conformation to the active state allows linkage to the transduction pathway (via the G-protein) and produces a biological response.
• a receptor may be stabilized in an active state by a ligand or a compound such as a drug.
• Recent discoveries, including but not exclusively limited to modifications to the amino acid sequence of the receptor provide means other than ligands or drugs to promote and stabilize the receptor in the active state conformation. These means effectively stabilize the receptor in an active state by simulating the effect of a ligand binding to the receptor.
• GPCRs G-protein coupled receptors
• At least four human and three rat splice variants have proven functional activity in pharmacological assays (Passani et al., Trends in Pharmacol. Sci. 2004, 25, 618-625).
• Rat and human histamine H3 -receptors also show constitutive activity which means that they can transduce a signal even in the absence of a ligand. Histamine H3 -receptors also function as heteroreceptors, modulating the release of a number of other transmitter substances including serotonin, acetylcholine, dopamine and noradrenaline (see: Brown et al. Prog. Neurobiol. 2001, 63, 637-672).
• the ligand functions as either an antagonist or inverse agonist (for reviews see: Leurs et al. Nat. Rev. Drug. Discov.
• Antagonists of H1R also known as antihistamines, have long been used to treat allergies such as hay fever (rhinitis). Although they are also widely prescribed as anti -pruritus medications, their efficacy, particularly for chronic skin diseases such as atopic dermatitis, has been a subject of debate.
• H4R has also recently been described to mediate itch. H4R deficient mice develop less severe pruritus compared to wild-type littermates, and the selective H4R receptor antagonist
• JNJ7777120 inhibited pruritus in several mouse pruritus models (Dunford, P.J., et al., J. Allergy Clin. Immunol. 2007, 119: 176-83).
• H3R inhibition does not reduce histamine-mediated pruritus. In fact, H3R inhibition has been linked to wakefulness and other related CNS mediated effects. Moreover, multiple groups (Br J Dermatol. 2003 Jul; 149 (1): 17-22 and Clin Exp Allergy. 2004 Mar;34 (3):456-9) have found that intradermal injection of H3R antagonists (i.e. thioperamide, iodophenpropit and clobenpropit) actually promote scratching behavior in mice. Therefore, prior to the present invention, existing reports suggested that H3R antagonism was not a viable treatment strategy for itch-associated disorders and, in fact, such treatment might actually be pruritogenic.
• H3R antagonists i.e. thioperamide, iodophenpropit and clobenpropit
• the present invention relates to the unexpected discovery that inhibition of the histamine 3 receptor (H3R) in an individual, such as by administration of a H3R antagonist, can reduce itching or pruritus.
• the present invention describes methods relating to screening assays performed with H3R for identifying H3R antagonists, for example antagonists or inverse agonists, useful for treating a condition characterized by itching, methods for using H3R antagonists in model systems, for example pruritus model systems, for determining efficacy or usefulness, methods for using H3R antagonists (e.g. partial antagonists, full antagonists, and inverse agonists) in the treatment of a condition characterized by itching, and compounds useful for the treatment of a condition characterized by itching, for example, in an individual.
• the individual is a human.
• the present invention provides a method of treating pruritus in a subject in need thereof by administering a therapeutically effective amount of a compound or agent that modulates the H3R.
• the compound or agent is an H3R antagonist.
• the H3R antagonist is a H3R inverse agonist.
• peripherally restricted antagonists of H3R are capable of mediating the inhibition of itch. Accordingly, screening for peripherally restricted antagonists (e.g.
• H3R antagonists for example H3R inverse agonists, are peripherally restricted and may be assayed or screened based on their inability or reduced ability to inhibit H3R in the CNS (for example, in the brain).
• H3R antagonists have been disclosed in the art. For example,
• H3R antagonists are disclosed in the following PCT Publications. Accordingly, one aspect of the present invention encompasses every combination of one or more H3R antagonist selected from the H3R antagonists disclosed in each of the PCT Publications and pharmaceutically acceptable salts, solvates and hydrates thereof:
• One aspect of the present invention relates to methods for treating pruritus in an individual comprising administering to the individual in need thereof a therapeutically effective amount of a H3R antagonist.
• One aspect of the present invention relates to use of a H3R antagonist in the manufacture of a medicament for treating pruritus.
• One aspect of the present invention relates to H3R antagonists for use in a method for treating pruritus.
• the invention disclosed herein is suitable for the treatment of pruritus that is associated with a disease or disorder selected from eczema, atopic eczematous dermatitis, seborrheic dermatitis, atopic dermatitis, contact dermatitis, irritant dermatitis, xerosis (dry skin), psoriasis, a fungal infection, athlete's foot, a yeast infection, diaper rash, vaginal itch, parasitic infections, parasitic infestations including scabies and lice, lichen planus, lichen simplex, lichen simplex chronicus, lichen sclerosis, itch secondary to medications, senile itch, uremia, idiopathic itch, itch associated with liver cirrhosis, itch associated with inflammation, itch associated with allergies, itch associated with cancer, itch associated with chemotherapy, itch associated with kidney disease, itch associated with haemodialysis, burns, scalds, sunburn, wound
• the invention disclosed herein utilizes a H3R antagonist, for example, a H3R antagonist from the genera described herein.
• the invention disclosed herein utilizes a H3R antagonist, for example, a H3R antagonist found in Table A.
• the H3R antagonist is peripherally restricted. In one embodiment, the H3R antagonist is applied topically to the site afflicted with itch. In another embodiment, the H3R antagonist is administered systemically. In one embodiment, the H3R antagonist is administered in the form of a pharmaceutical composition comprising a therapeutically effective amount of the H3R antagonist as the active ingredient, in admixture with a pharmaceutical carrier.
• the H3R antagonist pharmaceutical composition can be formulated in a form suitable for topical application such as a skin patch.
• the H3R antagonist is administered in conjunction with a pharmaceutically acceptable carrier.
• the invention features a method for identifying a compound capable of inhibiting itch in an individual, the method comprising administering a H3R antagonist, such as an antagonist or an inverse agonist, to a mammal for which reduction of itch may be evaluated, and evaluating whether the administration of the compound results in reduced itch.
• a H3R antagonist such as an antagonist or an inverse agonist
• H3R antagonists may be screened in established models for evaluating itch (i.e. animal, cellular, in vitro or in vivo).
• the invention features a method for identifying an antagonist (e.g. partial antagonists, full antagonists, and inverse agonists) of a H3R comprising: (a) contacting cells expressing the H3R in the presence of a known amount of histamine or surrogate thereof with a sample to be tested for the presence of a H3R antagonist; and (b) measuring at least one cellular function modulated by the binding of a ligand to the H3R, wherein the cellular function is selected from the group consisting of changes in intracellular second- messengerger levels, cell growth rates and hormone secretion; whereby the H3R antagonist in the sample is identified by measuring its effect on the cellular function compared to what would be measured in the absence of such antagonist.
• an antagonist e.g. partial antagonists, full antagonists, and inverse agonists
• the nucleotide sequence encoding human H3R polypeptide is given in SEQ ID NO: 1.
• the amino acid sequence of the encoded human H3R polypeptide is given in SEQ ID NO:2.
• One aspect of the present invention relates to methods for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, comprising the steps of:
• amino acids 2-445 of SEQ ID NO:2 with the proviso that the G protein-coupled receptor does not comprise the amino acid sequence of SEQ ID NO:2;
• test compound wherein the ability of the test compound to decrease the response in a pruritus model is indicative of the test compound being an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual.
• the G protein-coupled receptor comprises the amino acid sequence of SEQ ID NO:2.
• the variant of SEQ ID NO:2 is an allele of SEQ ID NO:2.
• the variant of SEQ ID NO:2 is an ortholog of SEQ ID NO:2. In certain embodiments, the variant of SEQ ID NO:2 is a mammalian ortholog of SEQ ID NO:2.
• the G protein-coupled receptor is recombinant.
• the method is a method for identifying compounds useful for treating a condition characterized by itch.
• the host cell is derived or obtained from a vertebrate.
• the vertebrate is a mammal. In certain embodiments, the vertebrate is a non-human vertebrate. In certain embodiments, the mammal is a non-human mammal.
• the invention additionally features a method for identifying compounds useful for treating a condition characterized by itch in an individual, optionally comprising steps (a) and (b) of the prior embodiments, and further comprising:
• step (c) optionally synthesizing a compound which inhibits functionality of the receptor in step (b);
• step (d) administering a compound which inhibits functionality of the receptor in step (b) to a vertebrate;
• test compound wherein the ability of the test compound to treat or prevent pruritus in the vertebrate is indicative of the test compound being a compound useful for treating a condition characterized by itch in an individual.
• One aspect of the present invention relates to methods for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, further comprising the step of: determining the chemical structure of the anti-pruritic agent. It is understood that this step can occur at any time after the step of determining the ability of the test compound to inhibit functionality of the G protein-coupled receptor (H3R) or the step of determining whether less of the complex (i.e., between the known ligand and the G protein-coupled receptor (H3R)) is formed in the presence of the test compound than in the absence of the test compound.
• this step can occur at any time after the step of determining the ability of the test compound to inhibit functionality of the G protein-coupled receptor (H3R) or the step of determining whether less of the complex (i.e., between the known ligand and the G protein-coupled receptor (H3R)) is formed in the presence of the test compound than in the absence of the test compound.
• step (a) the steps of contacting a test compound with a host cell or with membrane of a host cell comprising a G protein-coupled receptor (i.e., step (a)) and determining the ability of the test compound to inhibit functionality of the G protein-coupled receptor (i.e., step (b)) are not required.
• the determining comprises measuring a level of itch in the vertebrate.
• One aspect of the present invention relates to methods for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, comprising the steps of:
• test compound wherein the ability of the test compound to decrease the response in the pruritus model is indicative of the test compound being an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual.
• One aspect of the present invention relates to methods for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, comprising the steps of:
• test compound wherein the ability of the test compound to decrease the response in the pruritus model is indicative of the test compound being an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual.
• One aspect of the present invention relates to methods for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, comprising the steps of:
• test compound wherein the ability of the test compound to reduce the response in the animal is indicative of the test compound being an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual.
• One aspect of the present invention relates to methods for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, comprising the steps of:
• test compound wherein the ability of the test compound to decrease the response in the animal is indicative of the test compound being an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual.
• One aspect of the present invention relates to methods for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, comprising the steps of:
• test compound wherein the ability of the test compound to decrease the response in the animal is indicative of the test compound being an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual.
• One aspect of the present invention relates to methods for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, comprising the steps of:
• test compound wherein the ability of the test compound to decrease the response in the pruritus model is indicative of the test compound being an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual.
• One aspect of the present invention relates to methods for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, comprising the steps of:
• G protein-coupled receptor (a) contacting a G protein-coupled receptor with an optionally labeled known ligand to the receptor in the presence or absence of a test compound, wherein the G protein- coupled receptor comprises an amino acid sequence selected from the group consisting of:
• test compound wherein the ability of the test compound to decrease the response in the pruritus model is indicative of the test compound being an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual.
• optionally labeled known ligand refers to a known ligand that is optionally labeled, such as, a known ligand where one or more atoms are replaced or substituted by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature (i.e., naturally occurring).
• Suitable radionuclides that may be incorporated in the known ligand include but are not limited to 3 H (also written as T for tritium), n C, 13 C, 14 C, 13 N, 15 N, 15 0, 17 0, 18 0, 18 F, 35 S, 36 C1, 82 Br, 75 Br, 76 Br, 77 Br, 123 I, 124 1, 125 I, and 131 I.
• 3 H also written as T for tritium
• n C, 13 C, 14 C, 13 N, 15 N, 15 0, 17 0, 18 0, 18 F 35 S, 36 C1, 82 Br, 75 Br, 76 Br, 77 Br, 123 I, 124 1, 125 I, and 131 I.
• a know ligand that incorporates 3 H, 14 C, 35 S, 82 Br, 75 Br, 76 Br, 77 Br, 125 I, and 131 I will generally be most useful.
• One aspect of the present invention relates to methods for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, comprising the steps of:
• G protein-coupled receptor (a) contacting a G protein-coupled receptor with an optionally labeled known ligand to the receptor in the presence or absence of a test compound, wherein the G protein- coupled receptor comprises an amino acid sequence selected from the group consisting of:
• test compound wherein the ability of the test compound to decrease the response in the pruritus model is indicative of the test compound being an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual.
• One aspect of the present invention relates to methods for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, comprising the steps of:
• G protein-coupled receptor (a) contacting a G protein-coupled receptor with an optionally labeled known ligand to the receptor in the presence or absence of a test compound, wherein the G protein- coupled receptor comprises an amino acid sequence selected from the group consisting of:
• test compound wherein the ability of the test compound to decrease the response in the pruritus model is indicative of the test compound being an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual.
• One aspect of the present invention relates to methods for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, comprising the steps of:
• G protein-coupled receptor (a) contacting a G protein-coupled receptor with an optionally labeled known ligand to the receptor in the presence or absence of a test compound, wherein the G protein- coupled receptor comprises an amino acid sequence selected from the group consisting of:
• test compound wherein the ability of the test compound to decrease the response in the animal is indicative of the test compound being an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual.
• One aspect of the present invention relates to methods for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, comprising the steps of:
• G protein-coupled receptor (a) contacting a G protein-coupled receptor with an optionally labeled known ligand to the receptor in the presence or absence of a test compound, wherein the G protein- coupled receptor comprises an amino acid sequence selected from the group consisting of:
• test compound (d) observing a decreased response in a pruritus model test animal previously administered the test compound; wherein the ability of the test compound to decrease the response in the animal is indicative of the test compound being an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual.
• One aspect of the present invention relates to methods for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, comprising the steps of:
• G protein-coupled receptor (a) contacting a G protein-coupled receptor with an optionally labeled known ligand to the receptor in the presence or absence of a test compound, wherein the G protein- coupled receptor comprises an amino acid sequence selected from the group consisting of:
• test compound wherein the ability of the test compound to decrease the response in the pruritus model is indicative of the test compound being an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual.
• One aspect of the present invention relates to methods for identifying an anti-pruritic agent for treating a condition characterized by itch in an individual, comprising the steps of:
• H3R antagonist to decrease the response in said pruritus model is indicative of said H3R antagonist being an anti-pruritic agent useful for the treatment of said condition characterized by itch in said individual.
• two or more potential or actual H3R antagonists or anti-pruritic agents may be compared with each other in pruritus models. Such a comparison may be based, for example, on efficacy or brain to plasma distribution or any other aspect relevant to the H3R inhbitior or anti-pruritic agent(s) as described herein.
• One aspect of the present invention relates to methods for evaluating two or more H3R antagonists comprising the steps of:
• evaluating the two or more anti-pruritic agents by comparing the ability of each anti-pruritic agent to decrease a behavioral response to pruritus.
• One aspect of the present invention relates to methods for evaluating a H3R antagonist at two or more different concentrations comprising the steps of:
• H3R antagonist separately introducing or evaluating the H3R antagonist at two or more concentrations in a pruritus model
• evaluating further comprises selecting the anti-pruritic agent or concentration of anti-pruritic agent with the best efficacy.
• the G protein-coupled receptor is coupled to a G protein. In certain embodiments, activation of the G protein-coupled receptor decreases a level of intracellular cAMP. In certain embodiments, the G protein is Gi/o.
• the G protein-coupled receptor variant is a variant that is encoded by a polynucleotide that is amplifiable by polymerase chain reaction of SEQ ID NO:2 or an allele thereof.
• the G protein-coupled receptor encoded by a polynucleotide that is amplifiable by polymerase chain reaction is an allele of SEQ ID NO:2.
• the G protein-coupled receptor encoded by a polynucleotide that is amplifiable by polymerase chain reaction exhibits a detectable level of constitutive activity.
• the constitutive activity is for increasing a level of intracellular cAMP.
• the constitutive activity is for causing melanophore cells to undergo pigment dispersion.
• stringent hybridization conditions comprise hybridization at 42°C in a solution comprising 50% formamide, 5xSSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 g/mL denatured, sheared salmon sperm DNA, followed by washing at 65°C in a solution comprising O.lxSSC.
• Hybridization techniques are well known to the skilled artisan.
• the GPCR encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1 is SEQ ID NO:2 or an allele thereof.
• the GPCR encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1 is an allele of SEQ ID NO:2. In certain embodiments, the GPCR encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1 is an ortholog of SEQ ID NO:2. In some embodiments, the GPCR encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1 exhibits a detectable level of constitutive activity. In some embodiments, the constitutive activity is for increasing a level of intracellular cAMP. In some embodiments, the constitutive activity is for causing melanophore cells to undergo pigment dispersion.
• the G protein-coupled receptor is part of a fusion protein comprising a G protein.
• Techniques for making a GPCR:G fusion construct are well known to the skilled artisan (see, e.g., International Application WO2002/42461).
• the host cell comprises an expression vector, the expression vector comprising a polynucleotide encoding the G protein- coupled receptor.
• the expression vector is pCMV. This vector was deposited with the American Type Culture Collection (ATCC) on October 13, 1998 (10801 University Boulevard., Manassas, VA 20110-2209 USA) under the provisions of the Budapest Treaty for the International Recognition of the Deposit of
• Microorganisms for the Purpose of Patent Procedure The DNA was tested by the ATCC and determined to be viable.
• the ATCC has assigned the following deposit number to pCMV: ATCC #203351.
• Other suitable expression vectors will be readily apparent to those of ordinary skill in the art, and a wide variety of expression vectors are commercially available (e.g., from Clontech, Palo Alto, CA; Stratagene, La Jolla, CA; and Invitrogen, Carlsbad, CA).
• the activated G protein-coupled receptor is constitutively active.
• the activated G protein-coupled receptor is activated by a ligand.
• the receptor comprises the amino acid sequence of SEQ ID NO:2.
• the step determining the ability of the test compound to inhibit the functionality of the receptor is through the measurement of a level of a second messenger.
• the second messenger is selected from the group consisting of cyclic AMP (cAMP), cyclic GMP (cGMP), inositol 1,4,5-triphosphate ( ⁇ 3 ), diacylglycerol (DAG), MAP kinase activity, MAPK/ERK kinase kinase- 1 (MEKK1) activity, and Ca 2+ .
• cAMP cyclic AMP
• cGMP cyclic GMP
• DAG diacylglycerol
• MEKK1 MAPK/ERK kinase kinase- 1
• the second messenger is cAMP and the level of cAMP is decreased.
• the level of cAMP is decreased.
• the step determining is through the use of a Melanophore assay, or through the measurement of GTPyS binding to a membrane comprising said GPCR.
• the anti-pruritic agent is a H3R antagonist.
• the H3R antagonist is a H3R inverse agonist.
• the H3R antagonist or anti-pruritic agent is peripherally restricted.
• the H3R antagonist or anti-pruritic agent has a brain to plasma ratio of about 0.5 or less after administration in a pruritus model.
• the H3R antagonist or anti-pruritic agent has a brain to plasma ratio of about 0.2 or less after administration in a pruritus model.
• the H3R antagonist or anti-pruritic agent has a brain to plasma ratio of about 0.1 or less after administration in a pruritus model.
• the pruritus model is a vertebrate model.
• the vertebrate model is a mammalian model.
• the pruritus model is a primate model, a dog model, a guinea pig model, a mouse model, or a cat model.
• the pruritus model is selected from the group: pruritogen injection model, passive cutaneous anaphylaxis model, allergic pruritus model, and spontaneous pruritus model.
• the pruritus model is selected from the group: histamine induced pruritus model, DNP-Ovalbumin pruritus model, and DNFB pruritus model.
• the response to pruritus is a behavioral response.
• the individual is a non-human mammal.
• the individual is a human.
• One aspect of the present invention pertains to methods and processes, further comprising the step of subsequently admixing the anti-pruritic agent with a pharmaceutical carrier.
• One aspect of the present invention pertains to methods and processes, further comprising the step of subsequently admixing the anti-pruritic agent with a pharmaceutical carrier to form an anti-pruritic pharmaceutical composition.
• One aspect of the present invention pertains to methods and processes, further comprising the step of formulating the anti-pruritic pharmaceutical composition into a form suitable for oral application.
• One aspect of the present invention pertains to methods and processes, further comprising the step of formulating said anti-pruritic pharmaceutical composition into a form suitable for topical application.
• the anti-pruritic agent is subsequently admixed with a
• the anti-pruritic agent is subsequently formulated with a pharmaceutical carrier.
• the step determining the ability of the test compound to inhibit functionality of the receptor comprises comparing the functionality of the receptor in the presence and absence of the test compound and observing a decreased functionality in the presence of the test compound as compared to in the absence of the test compound.
• the host cell is a vertebrate cell. In some embodiments, the host cell is mammalian. In some embodiments, the mammalian host cell is selected from the group consisting of 293, 293T, CHO, MCB3901, and COS-7. In some embodiments, the host cell is melanophore. Other suitable host cells will be readily apparent to those of ordinary skill in the art, and a wide variety of cell lines are available from the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209.
• the determining is consistent with the G protein-coupled receptor being a Gs-coupled receptor.
• the determining is consistent with the G protein-coupled receptor being coupled through a promiscuous G protein, such as Gal5 or Gal6, to the phospholipase C pathway.
• a promiscuous G protein such as Gal5 or Gal6
• Promiscuous G proteins are well known to the skilled artisan (see, e.g., Offermanns et al., J Biol Chem (1995) 270: 15175-15180).
• the determining is consistent with the G protein-coupled receptor being coupled through a chimeric G protein, e.g. to the
• Chimeric G proteins are well known to the skilled artisan (see, e.g., Milligan et al., Trends in Pharmaceutical Sciences (1999) 20: 118-124; and WO2002/42461).
• the determining is through the measurement of a level of a second messenger.
• the determining is through the measurement of a level of a second messenger selected from the group consisting of cyclic AMP (cAMP), cyclic GMP (cGMP), inositol 1,4,5-triphosphate (IP 3 ), diacylglycerol (DAG), MAP kinase activity, MAPK/ERK kinase kinase- 1 (MEKK1) activity, and Ca 2+ .
• a second messenger is cAMP.
• a level of intracellular cAMP is increased.
• the determining is carried out using membrane comprising the G protein-coupled receptor.
• the determining is through the use of a melanophore assay. In certain embodiments, a level of pigment dispersion is increased.
• the determining is through the measurement of an activity mediated by increasing a level of intracellular cAMP.
• the determining is through CRE-Luc reporter assay. In certain embodiments, a level of luciferase activity is increased.
• the determining is through the measurement of GTPyS binding to membrane comprising the G protein-coupled receptor.
• the GTPyS is labeled with [ 35 S].
• the GTPyS binding to membrane comprising the GPCR is increased.
• the test compound is a small molecule.
• the test compound is a small molecule, with the proviso that the small molecule is not a polypeptide.
• the test compound is a small molecule, with the proviso that the small molecule is not an antibody or an antigen-binding fragment thereof.
• the test compound is a small molecule, with the proviso that the small molecule is not a lipid.
• the test compound is a small molecule, with the proviso that the small molecule is not a polypeptide or a lipid. In some embodiments, the test compound is a polypeptide. In some embodiments, the test compound is a polypeptide, with the proviso that the polypeptide is not an antibody or an antigen-binding fragment thereof. In some embodiments, the test compound is a lipid. In some embodiments, the test compound is not an antibody or an antigen-binding fragment thereof. In some embodiments, the test compound is an antibody or an antigen-binding fragment thereof. In some embodiments, the test compound is an anti-pruritic agent, a H3R antagonist, a H3R antagonist, or a H3R inverse agonist.
• the test compound is an anti-pruritic agent. In some embodiments, the test compound is a H3R antagonist. In some embodiments, the test compound is a H3R antagonist. In some embodiments, the test compound is a H3R inverse agonist.
• the known ligand is a ligand or agonist of an endogenous vertebrate, mammalian, or human H3R receptor. In certain embodiments, the known ligand is a known agonist of an endogenous vertebrate, mammalian, or human H3R receptor. In certain embodiments, the known ligand is a ligand or agonist of an endogenous human H3R receptor.
• the known ligand is an endogenous ligand of an endogenous vertebrate, mammalian, or human H3R receptor.
• the optionally labeled known ligand is a labeled known ligand.
• the labeled known ligand is a radiolabeled known ligand. Techniques for radiolabeling a compound, such as for labeling a known ligand of a G protein-coupled receptor of the invention, are well known to the skilled artisan. See, e.g., International Application
• the G protein-coupled receptor is part of a fusion protein comprising a G protein.
• Techniques for making a GPCR:G fusion construct are well known to the skilled artisan (see, e.g., International Application WO2002/42461).
• the determining is carried out using a host cell comprising the G protein-coupled receptor.
• the host cell comprises an expression vector, the expression vector comprising a polynucleotide encoding the GPCR.
• the expression vector is pCMV. This vector was deposited with the American Type Culture Collection (ATCC) on October 13, 1998 (10801 University Boulevard., Manassas, VA 20110-2209 USA) under the provisions of the Budapest Treaty for the International Recognition of the Deposit of
• Microorganisms for the Purpose of Patent Procedure The DNA was tested by the ATCC and determined to be viable.
• the ATCC has assigned the following deposit number to pCMV: ATCC #203351.
• Other suitable expression vectors will be readily apparent to those of ordinary skill in the art, and a wide variety of expression vectors are commercially available (e.g., from Clontech, Palo Alto, CA; Stratagene, La Jolla, CA; and Invitrogen, Carlsbad, CA).
• the host cell is a vertebrate cell. In some embodiments, the host cell is mammalian. In some embodiments, the mammalian host cell is selected from the group consisting of 293, 293T, CHO, MCB3901, and COS-7. In some embodiments, the host cell is melanophore. Other suitable host cells will be readily apparent to those of ordinary skill in the art, and a wide variety of cell lines are available from the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209.
• the determining is carried out using membrane comprising the G protein-coupled receptor.
• the method further comprises the step of optionally providing the name or structure of the compound useful for treating a condition characterized by itch in an individual.
• the invention features a method comprising, having identified a compound for treating a condition characterized by itch in an individual according to the invention, formulating the compound for treating a condition characterized by itch in an individual into a pharmaceutical composition.
• the invention features use of a G protein-coupled receptor to screen test compounds as anti-pruritic agents for treating a condition characterized by itch in an individual, wherein the G protein-coupled receptor comprises an amino acid sequence selected from the group consisting of:
• polynucleotide that is amplifiable by polymerase chain reaction (PCR) on a human DNA sample
• polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1;
• the screen is for an antagonist of the receptor.
• the screen is for a partial antagonist of the receptor.
• One aspect of the present invention pertains to methods of preparing a pharmaceutical composition
• a pharmaceutical composition comprising formulating an anti-pruritic agent with a pharmaceutically acceptable carrier; wherein the anti-pruritic agent is a H3R antagonist.
• One aspect of the present invention pertains to methods of preparing a pharmaceutical composition
• a pharmaceutical composition comprising formulating a H3R antagonist with a pharmaceutically acceptable carrier; wherein the H3R antagonist is an anti-pruritic agent.
• One aspect of the present invention pertains to methods of preparing a pharmaceutical composition
• a H3R antagonist having the effect of an anti-pruritic agent, said H3R antagonist having contacted in vitro with a host cell or with a membrane of a host cell comprising an activated G protein-coupled receptor, wherein said G protein-coupled receptor comprises an amino acid sequence selected from the group consisting of:
• One aspect of the present invention pertains to methods of preparing a pharmaceutical composition comprising an anti-pruritic agent having the effect of a H3R antagonist, said method comprising:
• test compound a biologically active fragment of any one of (i) to (v); wherein the ability of said test compound to inhibit the functionality of the activated G protein-coupled receptor in a host cell or with a membrane of a host cell is indicative of said test compound being an anti-pruritic agent;
• One aspect of the present invention pertains to methods of preparing a pharmaceutical composition comprising an anti-pruritic agent having the effect of a H3R antagonist, said method comprising:
• One aspect of the present invention pertains to methods of preparing a pharmaceutical composition comprising an anti-pruritic agent having the effect of a H3R antagonist, said method comprising: (a) contacting a test compound with a host cell or with a membrane of a host cell comprising an activated G protein-coupled receptor, wherein said G protein-coupled receptor comprises an amino acid sequence selected from the group consisting of:
• One aspect of the present invention pertains to methods of preparing a pharmaceutical composition comprising a H3R antagonist having the effect of an anti-pruritic agent, said H3R antagonist having obtained a decreased response for said H3R antagonist in a pruritus model, wherein the ability of said H3R antagonist to decrease the response in said pruritus model is indicative of said H3R antagonist being an anti-pruritic agent, said method comprising formulating said H3R antagonist having the effect of an anti-pruritic agent with a pharmaceutically acceptable carrier as a pharmaceutical composition.
• One aspect of the present invention pertains to methods of preparing a pharmaceutical composition comprising an anti-pruritic agent having the effect of a H3R antagonist, said method comprising:
• One aspect of the present invention relates to pharmaceutical compositions prepared by any of the methods or processes described herein.
• One aspect of the present invention relates to pharmaceutical compositions obtained by any of the methods or processes described herein.
• One aspect of the present invention relates to pharmaceutical compositions comprising a H3R antagonist and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is for the use in treating pruritus.
• One aspect of the present invention relates to pharmaceutical compositions comprising a H3R antagonist and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is for the use in treating pruritus.
• compositions comprising a H3R antagonist and a pharmaceutically acceptable carrier, wherein the H3R antagonist is identified using any of the methods or processes described herein.
• One aspect of the present invention relates to pharmaceutical compositions according to claim 12, wherein the H3R antagonist is identified using any of the methods or processes described herein and the pharmaceutical composition is for the use in treating pruritus.
• compositions comprising a
• H3R antagonist of the present invention and a pharmaceutically acceptable carrier.
• One aspect of the present invention pertains to methods for the treatment of pruritus in an individual, comprising administering to the individual in need thereof, a therapeutically effective amount of a H3R antagonist of the present invention or a pharmaceutical composition thereof.
• One aspect of the present invention pertains to the use of a H3R antagonist of the present invention or a pharmaceutical composition thereof in the manufacture of a medicament for the treatment of pruritus.
• One aspect of the present invention pertains to H3R antagonists of the present invention or a pharmaceutical composition thereof for use in a method of treatment of pruritus.
• One aspect of the present invention pertains to H3R antagonists for preparing a composition comprising admixing a H3R antagonist of the present invention and a
• Figure 1 shows the polynucleotide sequence of the human H3R.
• Figure 2 shows the polypeptide sequence of the human H3R.
• Figure 3 shows the polynucleotide sequence of the mouse H3R.
• Figure 4 shows the polypeptide sequence of the mouse H3R.
• Figure 5 shows that orally administered H3R antagonists are able to inhibit pruritus in a variety of pruritus animal models.
• Figure 5A shows that oral administration of Compound B and Compound C inhibit histamine (HA) induced pruritus.
• Figure 5B shows that oral administration of Compound A inhibits histamine-induced pruritus.
• Figure 5C shows that oral administration of Compound A inhibits serotonin-induced pruritus.
• Figure 5D shows that oral administration of Compound A inhibits Compound 48/80-induced pruritus.
• Figure 6 shows that topical application of Compound A inhibits histamine-induced itch.
• Figure 7 shows that administration of Compound A inhibits DNP-ovalbumin mediated allergic pruritus.
• Figure 8 shows that central administration of a H3R antagonist, Compound A, does not inhibit histamine-induced pruritus.
• Figure 8A shows ICV administration of Compound A increases cognitive function in mice.
• Figure 8B shows that ICV administration of Compound A is unable to inhibit histamine-induced pruritus. Suggesting that brain exposure of a H3R antagonist is not sufficient and that histamine induced pruritus is peripherally mediated.
• Figure 9 shows that administration of Compound 1 inhibits chronic DNFB -mediated allergic pruritus.
• Figure 10 shows the data from the administration of Compound 1 in the histamine- induced pruritus mouse model. The data shows that Compound 1 effectively inhibits histamine- induced pruritus in mice.
• Figure 11 shows the data from the administration of Compound 3 in the histamine- induced pruritus mouse model. The data shows that Compound 3 effectively inhibits histamine- induced pruritus in mice.
• Figure 12 shows a general synthesis of biaryl derivatives useful as intermediates in the preparation of compounds of the present invention (Formula (Va)).
• a mesylate derivative is coupled with an R 1 substituted pyrrolidine and subsequently converted to an aryl boronic acid by treatment of an aryl lithium intermediate with triisopropylborate followed by hydrolysis.
• the aryl boronic acid is coupled with an aryl bromide in the presence of a palladium catalyst to prepare the biaryl derivatives.
• Figure 13 shows general methods for preparing compounds of the present invention (Formula (Va)).
• Method A shows the use of carboxylic acids in the presence of a coupling agent to prepare compounds of the invention
• Method B shows the use of acid chlorides to prepare compounds of the invention, optionally in the presence of a base, such as an amine base.
• Figure 14 shows a general method for preparing compounds of the present invention (Formula (Va)).
• Method C shows the use of acid anhydrides in the presence of a base to prepare compounds of the invention.
• the present invention features methods of using H3R receptor to identify compounds useful for treating pruritus.
• Antagonists of H3R receptor are useful as therapeutic agents for treating a condition characterized by itch, such as pruritus.
• the present invention is based, at least in part, on the surprising discovery by Applicant that administration of a H3R antagonist to an individual, such as by oral administration, can act at the H3R receptor to reduce, prevent or treat pruritus and its symptoms. Accordingly, the invention further includes methods of evaluating known H3R antagonists in a variety of pruritus models for their usefulness in the treatment or prevention of pruritus.
• substituted indicates that at least one hydrogen atom of the chemical group is replaced by a non-hydrogen substituent or group, the non-hydrogen substituent or group can be monovalent or divalent. When the substituent or group is divalent, then it is understood that this group is further substituted with another substituent or group.
• a chemical group herein when a chemical group herein is "substituted" it may have up to the full valance of substitution; for example, a methyl group can be substituted by 1, 2, or 3 substituents, a methylene group can be substituted by 1 or 2 substituents, a phenyl group can be substituted by 1, 2, 3, 4, or 5 substituents, a naphthyl group can be substituted by 1, 2, 3, 4, 5, 6, or 7 substituents, and the like.
• substituted with one or more substituents refers to the substitution of a group with one substituent up to the total number of substituents physically allowed by the group. Further, when a group is substituted with more than one group they can be identical or they can be different.
• Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution. It is understood that the various tautomeric forms are within the scope of the compounds of the present invention.
• compounds of Formulae (la), (Ila), (Ilia), (IVa), (IVb), and (Va) and formulae related thereto may have one or more chiral centers and therefore can exist as enantiomers and or diastereoisomers.
• the invention is understood to extend to and embrace all such enantiomers, diastereoisomers and mixtures thereof, including but not limited to racemates.
• compounds of Formulae (la), (Ha), (Ilia), (IVa), (IVb), and (Va) and other formulae used throughout this disclosure represent all individual enantiomers and mixtures thereof, unless stated or shown otherwise.
• ligand shall mean a molecule (e.g., test compound) that specifically binds to a polypeptide, such as H3R.
• a ligand may be, for example, a polypeptide, a lipid, a small molecule, an antibody.
• An endogenous ligand is a ligand that is an endogenous, natural ligand for a native polypeptide, such as H3R.
• a ligand may be an "antagonist”, “agonist”, “partial agonist”, or “inverse agonist”, or the like. Exemplary antagonists of the H3R receptor are described herein.
• the H3R antagonist is a H3R inverse agonist.
• One aspect of the present invention encompasses every combination of one or more H3R receptor ligands selected from the H3R receptor ligands found in PCT published patent application WO2008/005338 and pharmaceutically acceptable salts, solvates, and hydrates thereof.
• H3R receptor ligands selected from compounds of Formula (la) and pharmaceutically acceptable salts, solvates, and hydrates thereof:
• R' andR 2 are each selected independently from the group consisting of H, C1-C6 acyl, Ci- C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 7 cycloalkyl, aryl, heterocyclyl, heteroaryl, aryl-Ci-C - alkylenyl, aryloxy-Ci-C 4 -alkylenyl, heteroaryl-Ci-C 4 -alkylenyl and heteroaryloxy-Ci-C 4 -alkylenyl, and each R 1 andR 2 is optionally substituted with 1, 2, 3, 4 or 5 substituents selected independently from the group consisting of Ci-C6 acyl, Ci-C6 acyloxy, C 2 -C 8 alkenyl, Ci-C6 alkoxy, Ci-C 8 alkyl, Ci-Cs alkylcarboxamide, C 2 -C 8 alkynyl, Ci
• R 1 and R 2 together with the nitrogen atom to which they are both bonded form a C 3 -C 7 heterocyclyl or a C5-C10 heterobicyclyl group each optionally substituted with 1, 2, 3, 4, 5 or 6 substituents selected independently from the group consisting of C1-C6 acyl, C1-C6 acyloxy, C 2 -C 8 alkenyl, C1-C6 alkoxy, Ci-C 8 alkyl, Ci-C 8 alkylcarboxamide, C 2 -C 8 alkynyl, Ci-C 8
• Ci-C 8 alkylsulfonamide Ci-C 8 alkylsulfinyl, Ci-C 8 alkylsulfonyl, Ci-C 8 alkylthio, Ci-C 8 alkylureyl, amino, Ci-C 8 alkylamino, C 2 -C 8 dialkylamino, carbo-Ci-C6-alkoxy, carboxamide, carboxy, cyano, C3-C7 cycloalkyl, C 2 -C 8 dialkylcarboxamide, C 2 -C 8 dialkylsulfonamide, halogen, C1-C6 haloalkoxy, C1-C6 haloalkyl, C1-C6 haloalkylsulfinyl, C1-C6 haloalkylsulfonyl, C1-C6 haloalkylthio, hydroxyl, thiol, nitro, oxo and sulfonamide; wherein each
• R 2 is selected independently from the group consisting of H, C1-C6 acyl, Ci-C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C3-C7 cycloalkyl, aryl, heterocyclyl, heteroaryl, aryl-Ci-C -alkylenyl, aryloxy-Ci-C -alkylenyl, heteroaryl-Ci-C -alkylenyl and heteroaryloxy-Ci-C -alkylenyl, and each R 2 is optionally substituted with 1, 2, 3, 4 or 5 substituents selected independently from the group consisting of Ci-C 6 acyl, Ci-C 6 acyloxy, C 2 -C 8 alkenyl, Ci-C 6 alkoxy, Ci-C 8 alkyl, Ci-C 8 alkylcarboxamide, C 2 -C 8 alkynyl, Ci-C 8 alkylsulfonamide
• R 1 and R 12 together with the atoms to which they are both bonded form a C6-C 8 heterocyclyl group optionally substituted with 1, 2, 3, 4 or 5 substituents selected independently from the group consisting of Ci-C6 acyl, Ci-C6 acyloxy, C 2 -C 8 alkenyl, Ci-C6 alkoxy, Ci-C 8 alkyl, Ci-Cs alkylcarboxamide, C 2 -C 8 alkynyl, Ci-C 8 alkylsulfonamide, Ci-C 8 alkylsulfinyl, Ci-C 8 alkylsulfonyl, Ci-C 8 alkylthio, Ci-C 8 alkylureyl, amino, aryl, Ci-C 8 alkylamino, C 2 -C 8 dialkylamino, carbo-Ci-C6-alkoxy, carboxamide, carboxy, cyano, C 3 -C 7 cycloalkyl, C 2 -C 8 dial
• J is -CH 2 CH 2 - or a l,2-C 3 -C 7 -cycloalkylenyl group, each optionally substituted with 1, 2, 3 or 4 substituents selected independently from the group consisting of C 1 -C3 alkyl, C 1 -C4 alkoxy, carboxy, cyano, C 1 -C3 haloalkyl, halogen, hydroxyl and oxo;
• R 3 , R 4 , R 5 , R 6 , R 7 , R 10 , R 11 and R 12 are each selected independently from the group consisting of H, C 1 -C6 acyl, C 1 -C6 acyloxy, C 2 -C 8 alkenyl, C 1 -C6 alkoxy, Ci-C 8 alkyl, Ci-C 8 alkylcarboxamide, C 2 -C 8 alkynyl, Ci-C 8 alkylsulfonamide, Ci-C 8 alkylsulfinyl, Ci-C 8 alkylsulfonyl, Ci-C 8 alkylthio, Ci-C 8 alkylureyl, amino, Ci-C 8 alkylamino, C 2 -C 8 dialkylamino, carbo-Ci-C 6 -alkoxy, carboxamide, carboxy, cyano, C3-C7 cycloalkyl, C 2 -C 8 dialkylcarboxamide, C 2 -
• R 8 and R 9 are each selected independently from the group consisting of H, Ci-C 8 alkyl, C 2 - C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 7 cycloalkyl, aryl, heterocyclyl, heteroaryl, aryl-Ci-C 4 -alkylenyl, aryloxy-Ci-C 4 -alkylenyl, heteroaryl-Ci-C 4 -alkylenyl and heteroaryloxy-Ci-C 4 -alkylenyl, and each R 8 and R 9 is optionally substituted with 1 , 2, 3, 4 or 5 substituents selected independently from the group consisting of Ci-C 6 acyl, Ci-C 6 acyloxy, C 2 -C 8 alkenyl, Ci-C 6 alkoxy, C C 8 alkyl, C C 8 alkylcarboxamide, C 2 -C 8 alkynyl, Ci-C 8 alkylsul
• R 8 and R 9 together with the nitrogen atom to which they are both bonded form a C 3 -C 7 heterocyclyl or a C5-C 10 heterobicyclyl group each optionally substituted with 1, 2, 3, 4, 5 or 6 substituents selected independently from the group consisting of C 1 -C6 acyl, C 1 -C6 acyloxy, C 2 -C 8 alkenyl, C 1 -C6 alkoxy, Ci-C 8 alkyl, Ci-C 8 alkylcarboxamide, C 2 -C 8 alkynyl, Ci-C 8
• alkylsulfonamide Ci-C 8 alkylsulfinyl, Ci-C 8 alkylsulfonyl, Ci-C 8 alkylthio, Ci-C 8 alkylureyl, amino, Ci-C 8 alkylamino, C 2 -C 8 dialkylamino, carbo-Ci-C6-alkoxy, carboxamide, carboxy, cyano, C3-C7 cycloalkyl, C 2 -C 8 dialkylcarboxamide, C 2 -C 8 dialkylsulfonamide, halogen, C 1 -C6 haloalkoxy, C 1 -C6 haloalkyl, C 1 -C6 haloalkylsulfinyl, C 1 -C6 haloalkylsulfonyl, C 1 -C6 haloalkylthio, hydroxyl, thiol, nitro, oxo and sulfonamide; provided that
• One aspect of the present invention encompasses every combination of one or more H3R receptor ligands selected from the H3R receptor ligands found in PCT published patent application WO2008/048609 and pharmaceutically acceptable salts, solvates, and hydrates thereof.
• H3R receptor ligands selected from compounds of Formula (Ha) and pharmaceutically acceptable salts, solvates, and hydrates thereof:
• R 1 is selected from the group consisting of H, C 1 -C6 acyl, C 1 -C6 acyloxy, C 2 -C 8 alkenyl, C 1 -C6 alkoxy, Ci-C 8 alkyl, Ci-C 8 alkylcarboxamide, C 2 -C 8 alkynyl, Ci-C 8 alkylsulfonamide, Ci-C 8 alkylsulfinyl, Ci-C 8 alkylsulfonyl, Ci-C 8 alkylthio, Ci-C 8 alkylureyl, amino, Ci-C 8 alkylamino, C 2 - C 8 dialkylamino, carbo-Ci-C6-alkoxy, carboxamide, carboxy, cyano, C 3 -C 7 cycloalkyl, C 2 -C 8 dialkylcarboxamide, C 2 -C 8 dialkylsulfonamide, halogen, C 1 -C6 haloalkoxy, C
• Ci-C 8 alkyl Ci-C 8 alkylcarboxamide, C 2 -C 8 alkynyl, Ci-C 8 alkylsulfonamide, Ci-C 8 alkylsulfinyl, Ci-C 8 alkylsulfonyl, Ci-C 8 alkylthio, Ci-C 8 alkylureyl, amino, Ci-C 8 alkylamino, C 2 -
• R 1 together with the W-S0 2 group and the ring atom to which the W-S0 2 group is bonded form a C5-C7 heterocyclic ring with Ring A whereby the C5-C7 heterocyclic ring and Ring A share two adjacent ring atoms, and the C5-C7 heterocyclic ring is optionally substituted with 1, 2, 3 or 4 substituents selected independently from the group consisting of C 1 -C6 acyl, C 1 -C6 acyloxy, C 2 -C 8 alkenyl, C 1 -C6 alkoxy, Ci-C 8 alkyl, Ci-C 8 alkylcarboxamide, C 2 -C 8 alkynyl, Ci-C 8
• alkylsulfonamide Ci-C 8 alkylsulfinyl, Ci-C 8 alkylsulfonyl, Ci-C 8 alkylthio, Ci-C 8 alkylureyl, amino, Ci-C 8 alkylamino, C 2 -C 8 dialkylamino, carbo-Ci-C6-alkoxy, carboxamide, carboxy, cyano, C3-C7 cycloalkyl, C 2 -C 8 dialkylcarboxamide, C 2 -C 8 dialkylsulfonamide, halogen, C 1 -C6 haloalkoxy, C 1 -C6 haloalkyl, C 1 -C6 haloalkylsulfinyl, C 1 -C6 haloalkylsulfonyl, C 1 -C6 haloalkylthio, hydroxyl, thiol, nitro, oxo and sulfonamide;
• W is C 1 -C4 alkylene, C 2 -C 4 alkenylene, C3-C7 cycloalkylene, C3-C7 heterocyclylene or phenylene, each optionally substituted with 1, 2, 3, 4, 5, 6, 7 or 8 substituents selected
• Ring A is 1,3-phenylene or 1,4-phenylene, each substituted with R 12 , R 13 , R 14 and R 15 , wherein R 12 , R 13 , R 14 and R 15 are each selected independently from the group consisting of H, C 1 -C6 acyl, Ci-C 6 acyloxy, C 2 -C 8 alkenyl, Ci-C 6 alkoxy, Ci-C 8 alkyl, Ci-C 8 alkylcarboxamide, C 2 -C 8 alkynyl, C C 8 alkylsulfonamide, C C 8 alkylsulfinyl, C C 8 alkylsulfonyl, C C 8 alkylthio, C C 8 alkylureyl, amino, Ci-C 8 alkylamino, C 2 -C 8 dialkylamino, carbo-Ci-C6-alkoxy, carboxamide, carboxy, cyano, C3-C7 cycloalkyl, C 2 -C
• Ring A is a 6-membered heteroarylene or a 5-membered heteroarylene, each optionally substituted with R 16 , R 17 and R 18 , wherein R 16 , R 17 and R 18 are each selected independently from the group consisting of Ci-C 6 acyl, Ci-C 6 acyloxy, C 2 -C 8 alkenyl, Ci-C 6 alkoxy, C C 8 alkyl, C C 8 alkylcarboxamide, C 2 -C 8 alkynyl, Ci-C 8 alkylsulfonamide, Ci-C 8 alkylsulfinyl, Ci-C 8 alkylsulfonyl, Ci-C 8 alkylthio, Ci-C 8 alkylureyl, amino, Ci-C 8 alkylamino, C 2 -C 8 dialkylamino, carbo-Ci-C 6 - alkoxy, carboxamide, carboxy, cyano, C3-C7 cycloalkyl, C 2 -C
• R 2 , R 3 , R 4 andR 5 are each selected independently from the group consisting of H, C 1 -C6 acyl, C 1 -C6 acyloxy, C 2 -C 8 alkenyl, C 1 -C6 alkoxy, Ci-C 8 alkyl, Ci-C 8 alkylcarboxamide, C 2 -C 8 alkynyl, Ci-C 8 alkylsulfonamide, Ci-C 8 alkylsulfinyl, Ci-C 8 alkylsulfonyl, Ci-C 8 alkylthio, Ci-C 8 alkylureyl, amino, Ci-C 8 alkylamino, C 2 -C 8 dialkylamino, carbo-Ci-C6-alkoxy, carboxamide, carboxy, cyano, C3-C7 cycloalkyl, C 2 -C 8 dialkylcarboxamide, C 2 -C 8 dialkylsulfonamide, halogen, C 1
• R 6 , R 7 , R 8 andR 9 are each selected independently from the group consisting of H, C 1 -C3 alkyl, C 1 -C4 alkoxy, carboxy, cyano, C 1 -C3 haloalkyl, halogen and hydroxyl; and
• R 10 and R 11 together with the nitrogen atom to which they are both bonded form 2-methyl- pyrrolidin-l-yl;
• Ring B and the sulfur of the R 1 -W-S(0)2- group are not bonded to adjacent ring atoms of Ring A;
• Ring A is 1,3-phenylene or 1,4-phenylene
• W is C3-C7 heterocyclylene
• the ring atom of W that is directly bonded to the sulfur of the R 1 -W-S(0)2- group is other than nitrogen.
• H3R receptor ligand selected from (R)- 1 - ⁇ 2- [4'-(3-methoxy-propane- 1 -sulfonyl)-biphenyl-4-yl] -ethyl ⁇ -2-methyl -pyrrolidine and pharmaceutically acceptable salts, solvates, and hydrates thereof.
• H3R receptor ligands selected from the groups consisting of:
• One aspect of the present invention encompasses every combination of one or more H3R receptor ligands selected from the H3R receptor ligands found in PCT published patent application WO2009/058300 and pharmaceutically acceptable salts, solvates, and hydrates thereof.
• H3R receptor ligands selected from compounds of Formula (Ilia) and pharmaceutically acceptable salts, solvates, and hydrates thereof:
• ring A is heterocyclyl optionally substituted with one, two or three substituents selected from C 1 -C6 alkyl and oxo; wherein each Ci-C6 alkyl is optionally substituted with a Ci-C6 alkoxy substituent;
• R 1 is H, C 1 -C6 alkoxy, Ci-C6 alkyl or halogen
• R 2 is H, C 1 -C6 alkoxy, Ci-C6 alkyl or halogen
• R 3 is H, C 1 -C6 alkoxy, Ci-C6 alkyl or halogen
• R 4 is H or Ci-C 4 alkyl
• n 0, 1 or 2.
• One aspect of the present invention encompasses every combination of one or more H3R receptor ligands selected from the H3R receptor ligands found in PCT published patent application WO2009/ 105206 pharmaceutically acceptable salts, solvates, and hydrates thereof.
• H3R receptor ligands selected from compounds of Formula (IVa) and pharmaceutically acceptable salts, solvates, and hydrates thereof:
• R 1 is H or C 1 -C4 alkyl
• R 2 is H or halogen
• R 3 is H, C 1 -C4 alkyl or C 3 -C6 cycloalkyl, and R 4 is H; or R 3 and R 4 together with the atom to which they are both bonded form a C 3 -C6 cycloalkyl;
• R 5 is selected from: C 1 -C6 alkyl, aryl, C 3 -C6 cycloalkyl, heteroaryl and heterocyclyl; each of which is optionally substituted with one or more substituents selected from: C 1 -C6 alkoxy, halogen, heterocyclyl and hydroxyl;
• R 6 , R 7 and R 8 are each independently selected from: H, Ci-Ce alkoxy, C 1 -C6 alkyl, amino, halogen, heterocyclyl and hydroxyl;
• n 0 or 1
• n 1 or 2;
• V is CH 2 , O or absent.
• H3R receptor ligands selected from compounds of Formula (IVb) and pharmaceutically acceptable salts, solvates, and hydrates thereof:
• R 5 is selected from: methyl, cyclopropyl, tetrahydropyran-4-yl, methoxymethyl, 2- methoxyethyl, 3-methoxypropyl, hydroxymethyl, 2-hydroxyethyl, tetrahydropyran-4-ylmethyl, 2,2- difluorocyclopropyl, 4-methoxyphenyl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, pyrimidin-5-yl, 6- hydroxypyridin-3-yl and 2-hydroxypyridin-4-yl.
• Compound A (R)-2-hydroxy- l-(6-(4-(2-(2-methylpyrrolidin-l- yl)ethyl)phenyl)-3,4-dihydroisoquinolin-2(l//)-yl)ethanone (referred to as Compound A herein), see Compound 10 in PCT publication WO2009/105206.
• H3R receptor ligands selected from (R)-2-hydroxy- 1 -(6-(4-(2-(2-methylpyrrolidin- 1 -yl)ethyl)phenyl)-3,4-dihydroisoquinolin- 2(l//)-yl)ethanone and pharmaceutically acceptable salts, solvates, and hydrates thereof.
• One aspect of the present invention encompasses, inter alia, certain isoquinoline derivatives selected from compounds of Formula (Va) and pharmaceutically acceptable salts, solvates, and hydrates thereof:
• n, and p are each independently 0 or 1;
• X is selected from the group: -S-, -0-, C3-C6 cycloalkylene, and CR C R C ;
• R 1 is H or C 1 -C4 alkyl
• R 2 is selected from the group: C 1 -C4 alkoxycarbonyl, carboxyl, and l/i-tetrazol-5-yl; and each R , R b , and R c is independently selected from the group: H, C 1 -C3 alkyl, amino, halogen, and hydroxyl.
• One aspect of the present invention encompasses every combination of one or more H3R inhibitors selected from the following H3R inhibitors and pharmaceutically acceptable salts, solvates, and hydrates thereof:
• One aspect of the present invention encompasses every combination of one or more H3R inhibitors selected from the H3R inhibitors in Table A and pharmaceutically acceptable salts, solvates, and hydrates thereof.
• H3R receptor ligands selected from 6-[(3-cyclobutyl-2,3,4,5-tetrahydro-lH-3-benzazepin-7-yl)oxy]-N-methyl-3- pyridinecarboxamide and pharmaceutically acceptable salts, solvates, and hydrates thereof.
• H3R receptor ligands selected from 6-[(3-cyclobutyl-2,3,4,5-tetrahydro-lH-3-benzazepin-7-yl)oxy]-N-methyl-3- pyridinecarboxamide hydrochloride and solvates and hydrates thereof.
• the chemical structure for 4-(3-(4-(piperidin-l-yl)but-l-ynyl)benzyl)morpholine (referred to as Compound C herein) is as follows:
• H3R receptor ligands selected from 4-(3-(4-(piperidin-l-yl)but-l-ynyl)benzyl)morpholine and pharmaceutically acceptable salts, solvates, and hydrates thereof.
• antagonist includes partial antagonists, full antagonists, and inverse agonists, and shall mean any organic or inorganic molecule that does not activate the intracellular response initiated by the active form of the receptor, and can thereby inhibit the intracellular responses of agonists and/or partial agonists. It is understood that an "antagonist” as defined herein can bind to the same active site as an agonist or the antagonist bind at a different site provided that the molecule is capable of inhibiting the response of a GPCR, such as, an allosteric antagonist. Another example includes nucleic acid molecules capable of interfering with the GPCR. Certainly, a H3R antagonist would include any anti-pruritic agent capable of inhibiting a response of the H3R. Inverse agonists are specific examples of H3R antagonists.
• anti-pruritic agent shall mean an agent capable of treating one or more aspects of pruritus. Agents may include, for example, small molecules, large molecules, nucleic acids, synthetic nucleic acid analogs, polypeptide(s), antibodies, and other biologies. H3R antagonists and inverse agonists are examples of anti-pruritic agents.
• agonist shall mean an agent (e.g., ligand, test compound) that by virtue of binding to a GPCR activates the GPCR so as to elicit an intracellular response mediated by the GPCR.
• partial agonist shall mean an agent (e.g., ligand, test compound) that by virtue of binding to a GPCR activates the GPCR so as to elicit an intracellular response mediated by the GPCR, albeit to a lesser extent or degree than does a full agonist.
• H3R antagonist refers to a compound that binds to H3R receptor and acts as an antagonist as defined herein.
• the H3R antagonist is a selective antagonist.
• the H3R antagonist has selectivity for H3R that is at least 10 fold greater than its selectivity for any one or more receptors selected from: HIR, H2R and H4R.
• the H3R antagonist has a selectivity for H3R over HIR of at least about 10- fold.
• the H3R antagonist has a selectivity for H3R over H2R of at least about 10-fold.
• the H3R antagonist has a selectivity for H3R over H4R of at least about 10-fold.
• the H3R antagonist has a selectivity for H3R over HIR, H2R, or H4R of at least about 10 fold. In various embodiments the H3R antagonist has in IC50 (H3R) of less than about 1 ⁇ , less than about 100 nM, less than about 10 nM or less than about 1 nM. In various embodiments the H3R antagonist has in IC50 (H3R) of about 1 ⁇ or less, about 100 nM or less, about 10 nM or less, or about 1 nM or less. In one embodiment, the H3R antagonist is an orally active H3R antagonist. In one embodiment, the H3R antagonist is peripherally restricted. In some embodiments, the H3R antagonist is an antagonist of the human
• the H3R antagonist is administered orally and has an IC50 (H3R) of about 100 nM or less. In some embodiments, the H3R antagonist is applied topically and has an IC50 (H3R) of about 100 nM or less. In some embodiments, the H3R antagonist is in an amount sufficient to reduce itch in a human.
• inverse agonist shall mean an agent (e.g., ligand, test compound) which binds to a GPCR and which inhibits the baseline intracellular response initiated by the active form of the receptor below the normal base level activity which is observed in the absence of an agonist or partial agonist.
• H3R inverse agonist refers to a compound that binds to H3R receptor and acts as an inverse agonist.
• the H3R inverse agonist is a selective inverse agonist.
• the H3R inverse agonist has selectivity for H3R that is at least 10 fold greater than its selectivity for any one or more receptors selected from: HIR, H2R and H4R.
• the H3R inverse agonist has a selectivity for H3R over HIR of at least about 10-fold.
• the H3R inverse agonist has a selectivity for H3R over H2R of at least about 10-fold.
• the H3R inverse agonist has a selectivity for H3R over H4R of at least about 10-fold. In one embodiments, the H3R inverse agonist has a selectivity for H3R over HIR, H2R, or H4R of at least about 10 fold. In various embodiments the H3R inverse agonist has in IC50 (H3R) of less than about ⁇ , less than about ⁇ , less than about ⁇ or less than about InM. In various embodiments the H3R inverse agonist has in IC50 (H3R) of about 1 ⁇ or less, about 100 nM or less, about 10 nM or less, or about 1 nM or less.
• the H3R inverse agonist is an orally active H3R inverse agonist. In one embodiment, the H3R inverse agonist is peripherally restricted. In some embodiments, the H3R inverse agonist is an inverse agonist of the human H3R. In some embodiments, the H3R inverse agonist is orally active and has an IC50 of less than about 100 nM. In some embodiments, the H3R inverse agonist is administered orally and has an IC50 (H3R) of about 100 nM or less. In some embodiments, the H3R inverse agonist is applied topically and has an IC50 (H3R) of about 100 nM or less. In some embodiments, the H3R inverse agonist is in an amount sufficient to reduce itch in a human.
• the individual is a fish.
• the individual is an amphibian.
• the individual is a reptile. In certain embodiments, the individual is a bird. In certain embodiments, the individual is a mammal. In certain embodiments, the individual is a mouse, a rat, a hamster, a rabbit, a pig, a dog, a cat, a horse, a cow, a sheep, a goat, a non-human primate, or a human (which may be included in embodiments of the invention individually or in any combination). In certain embodiments, the individual is a horse. In certain embodiments, the individual is a dog or a cat.
• the individual is a human companion animal (such as a dog, a cat, etc.), a farm animal (such as a cow, a sheep, a goat, a pig, a chicken, etc.), a sports animal (such as a horse, a dog, etc.), a beast of burden (such as a mule, a camel, etc.) or an exotic animal (such as an animal found in a zoo, etc.), which may be included in embodiments of the invention individually or in any combination.
• the individual is a non-human mammal.
• the individual is a non-human primate (such as a rhesus monkey, a chimpanzee, etc.).
• the individual is a human.
• treating and “treatment” as used herein refers to a judgment made by a caregiver (e.g. physician, nurse, nurse practitioner in the case of humans; veterinarian in the case of non-human vertebrates, and in particular embodiment non-human mammals) that an individual requires or will benefit from treatment.
• a caregiver e.g. physician, nurse, nurse practitioner in the case of humans; veterinarian in the case of non-human vertebrates, and in particular embodiment non-human mammals
• terapéuticaally effective amount refers to the amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue, system, animal, individual or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes one or more of the following: (1) Preventing the disease; for example, preventing a disease, condition or disorder in an individual that may be predisposed to the disease, condition or disorder but does not yet experience or display the pathology or symptomatology of the disease,
• Inhibiting the disease for example, inhibiting a disease, condition or disorder in an individual that is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., arresting further development of the pathology and/or symptomatology), and
• Ameliorating the disease for example, ameliorating a disease, condition or disorder in an individual that is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., reversing the pathology and/or symptomatology).
• therapeutic efficacy refers to elicitation of the biological or medicinal response in a tissue, system, animal, individual or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes one or more of the following:
• Preventing the disease for example, preventing a disease, condition or disorder in an individual that may be predisposed to the disease, condition or disorder but does not yet experience or display the pathology or symptomatology of the disease,
• Inhibiting the disease for example, inhibiting a disease, condition or disorder in an individual that is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., arresting further development of the pathology and/or symptomatology), and
• Ameliorating the disease for example, ameliorating a disease, condition or disorder in an individual that is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., reversing the pathology and/or symptomatology).
• composition shall mean a material comprising at least one component.
• active ingredient shall mean any component that provides pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of a disease.
• composition shall mean a composition comprising at least one active ingredient, whereby the composition is amenable to investigation and treatment in a mammal.
• pharmaceutically acceptable it is meant that the carrier, vehicle, diluent, excipients, and/or salt must be compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof.
• drug form shall mean the physical form in which a drug is produced and dispensed, such as a tablet, capsule, or an injectable.
• endogenous shall mean a material that an individual naturally produces.
• contact shall mean bringing at least two moieties together.
• modulate shall be taken to refer to an increase or decrease in the amount, quality, or effect of a particular activity, function or molecule.
• agonists, partial agonists, inverse agonists, and antagonists of a G protein-coupled receptor are modulators of the receptor.
• small molecule shall be taken to mean a compound having a molecular weight of less than about 10,000 grams per mole, including a peptide, peptidomimetic, amino acid, amino acid analogue, polynucleotide, polynucleotide analogue, nucleotide, nucleotide analogue, organic compound or inorganic compound (i.e. including a heterorganic compound or organometallic compound), and salts, esters and other pharmaceutically acceptable forms thereof.
• small molecules are organic or inorganic compounds having a molecular weight of less than about 5,000 grams per mole.
• small molecules are organic or inorganic compounds having molecular weight of less than about 1,000 grams per mole.
• small molecules are organic or inorganic compounds having a molecular weight of less than about 800 grams per mole. In certain embodiments, small molecules are organic or inorganic compounds having a molecular weight of less than about 600 grams per mole. In certain embodiments, small molecules are organic or inorganic compounds having a molecular weight of less than about 500 grams per mole.
• itch refers to the sensation that elicits a reflex response to scratch. Itch can be a symptom of a disease, disorder or infection, or itch can arise spontaneously, without an underlying or identifiable physiological cause, known as idiopathic pruritus.
• control of itch includes reducing, alleviating, relieving and numbing the sensation of itch. Control of itch also includes reducing the desire to scratch.
• in need of treatment and the term “in need thereof” when referring to treatment are used interchangeably and refer to a judgment made by a caregiver (e.g. physician, nurse, nurse practitioner, etc. in the case of humans; veterinarian in the case of animals, including non-human mammals) that an individual or animal requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of a caregiver' s expertise, but that includes the knowledge that the individual or animal is ill, or will become ill, as the result of a disease, condition or disorder that is treatable by the compounds of the invention.
• a caregiver e.g. physician, nurse, nurse practitioner, etc. in the case of humans; veterinarian in the case of animals, including non-human mammals
• the compounds of the invention can be used in a protective or preventive manner; or compounds of the invention can be used to alleviate, inhibit or ameliorate the disease, condition or disorder and refers to all aspects of control of itching including prophylaxis and therapy.
• Control of itch includes reducing, alleviating, relieving and numbing the sensation of itch.
• Control of itch also includes reducing the desire to scratch.
• antibody refers to immunoglobulin molecules and
• immunologically active portions of immunoglobulin molecules i.e., molecules that contain an antigen binding site that immunospecifically bind an antigen.
• the terms also refers to antibodies comprised of two immunoglobulin heavy chains and two immunoglobulin light chains as well as a variety of forms besides antibodies; including, for example, Fv, Fab, and F(ab)'2 as well as bifunctional hybrid antibodies (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)) and single chains (e.g., Huston et al., Proc. Natl. Acad. Sci.
• polypeptide shall refer to a polymer of amino acids without regard to the length of the polymer.
• peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term also does not specify or exclude post-expression modifications of polypeptides.
• polypeptides that include the covalent attachment of glycosyl groups, acetyl groups, phosphate groups, lipid groups and the like are expressly encompassed by the term polypeptide.
• antibody is intended herein to encompass monoclonal antibody and polyclonal antibody.
• second messenger shall mean an intracellular response produced as a result of receptor activation.
• a second messenger can include, for example, inositol 1,4,5-triphosphate (IP 3 ), diacylglycerol (DAG), cyclic AMP (cAMP), cyclic GMP (cGMP), MAP kinase activity, MAPK/ERK kinase kinase- 1 (MEKK1) activity, and Ca 2+ .
• Second messenger response can be measured for a determination of receptor activation.
• receptor functionality shall refer to the normal operation of a receptor to receive a stimulus and moderate an effect in the cell, including, but not limited to regulating gene transcription, regulating the influx or efflux of ions, effecting a catalytic reaction, and/or modulating activity through G-proteins, such as eliciting a second messenger response.
• “functionality of the receptor” shall mean that a response or a functionality of the receptor is increased in the presence of a compound as opposed to in the absence of the compound.
• inhibitor or “inhibiting,” in relationship to the term “response” or “functionality of the receptor” shall mean that a response a functionality of the receptor is decreased or prevented in the presence of a compound as opposed to in the absence of the compound.
• compound efficacy shall mean a measurement of the ability of a compound to inhibit or stimulate receptor functionality, as opposed to receptor binding affinity.
• the compound efficacy of different compounds may be evaluated with respect to each other or some other standard.
• test compound used interchangeably herein with “candidate compound,” shall mean a molecule (for example, and not limitation, a chemical compound) which is amenable to a screening technique.
• a test compound is an anti-pruritic agent.
• a test compound is a H3R antagonist.
• a test compound is a H3R inverse agonist.
• directly identifying or “directly identified”, in relationship to the phrase “test compound,” shall mean the screening of a compound against a G protein-coupled receptor in the absence of a known ligand (e.g., a known agonist) to the G protein-coupled receptor.
• a known ligand e.g., a known agonist
• the term "pruritus model” shall mean an in vivo or in vitro model system which displays at least one aspect or element of pruritus and is amenable to or allows the evaluation of anti-pruritic agents.
• the pruritus model is mammalian in origin.
• peripherally restricted shall refer to a compound or agent that is restricted in some manner from crossing the blood brain barrier.
• a peripherally restricted compound or agent may be less than 100% restricted to the periphery.
• a peripherally restricted compound may be further referred to by a braimplasma ratio which will be less than 1 in at least one species.
• H3R Histamine H3 Receptors
• the human histamine H3 receptor (GenBank accession number: AF140538) is described in Figure 1 and Figure 2, and SEQ ID NO: 1 and SEQ ID NO:2 (Lovenberg, T.W., Roland, B.L., Wilson, S.J., Jiang, X., Pyati, J., Huvar, A., Jackson, M.R. and Erlander, M.G., Cloning and functional expression of the human histamine H3 receptor. Mol. Pharmacol. 55 (6), 1101-1107 (1999)).
• the human histamine H3 receptor gene contains three introns and 4 exons. Through alternative splicing, six receptor variants are known to be created.
• the full-length receptor and two additional variants are known to bind histamine and other H3 agonists (Wellendorph P, Goodman MW, Burstein ES, Nash NR, Brann MR, Weiner DM. Molecular cloning and pharmacology of functionally distinct isoforms of the human histamine H(3) receptor. Neuropharmacology.
• mouse histamine H3 receptor (GeneBank accession number: AY142145) is described in Figure 3 and Figure 4 and SEQ ID NOS: 3 and 4 (Chen, J., Liu, C. and Lovenberg, T. W. Molecular and pharmacological characterization of the mouse histamine H(3) receptor. Eur J Pharmacol 467 (1-3), 57-65 (2003)).
• H3R Use of species variants of H3R are envisioned to be within the scope of the invention. Allelic variants of H3R of SEQ ID NO:2 are envisioned to be within the scope of the invention. Human H3R is envisioned to be within the scope of the invention.
• a variant which is a vertebrate ortholog of human H3R of SEQ ID NO:2 is envisioned to be within the scope of the invention.
• a variant which is a mammalian ortholog of human H3R of SEQ ID NO:2 is envisioned to be within the scope of the invention.
• mouse H3R, rat H3R, hamster H3R, dog H3R, and non-human primate H3R are envisioned to be within the scope of the invention.
• the variant of SEQ ID NO:2 is a GPCR.
• the GPCR comprises the amino acids 1-445 of SEQ ID NO:2.
• the GPCR comprises the amino acids 2-445 of SEQ ID NO:2.
• the GPCR comprises the amino acids 2-445 of SEQ ID NO:2, with the proviso that the GPCR does not comprise the amino acid sequence of SEQ ID NO: 2.
• the GPCR comprises the amino acid sequence of a GPCR encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: l.
• the GPCR comprises a variant of SEQ ID NO: 2.
• a variant of SEQ ID NO:2 having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO:2 is envisioned to be within the scope of the invention.
• the variant of SEQ ID NO:2 having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO:2 is a GPCR.
• a variant GPCR that may be used in the subject methods has an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, of at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO:2.
• a variant GPCR having, for example, 95% "identity" to SEQ ID NO:2 is meant that the amino acid sequence of the variant is identical to amino acids 1-335 of SEQ ID NO:2 except that it may include up to five amino acid alterations per each 100 amino acids of SEQ ID NO:2.
• amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:2 up to 5% (5 of 100) of the amino acid residues in the sequence may be inserted, deleted, or substituted with another amino acid compared with amino acids 1-617 of SEQ ID NO: 2. These alternations may occur at the amino or carboxy termini or anywhere between those terminal positions, interspersed either subjectly among residues in the sequence or in one or more contiguous groups within the sequence.
• a variant GPCR that may be used in the subject methods is a GPCR encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1.
• the GPCR encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1 is an endogenous GPCR.
• the GPCR encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1 and that is an endogenous GPCR is a mammalian endogenous GPCR.
• the GPCR encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1 is SEQ ID NO:2 or an allele thereof. In certain embodiments, the GPCR encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1 is an allele of SEQ ID NO:2. In certain embodiments, the GPCR encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1 is an ortholog of SEQ ID NO:2.
• the GPCR is screened in antagonist mode.
• a GPCR of the invention forms part of a fusion protein with a G protein.
• a preferred method for determining the best overall match between a query sequence e.g., the amino acid sequence of SEQ ID NO:2
• a sequence to be interrogated also referred to as a global sequence alignment
• FASTDB computer program based on the algorithm of Brutlag et al. (Comp App Biosci (1990) 6:237-245).
• sequence alignment the query and interrogated sequences are both amino acid sequences.
• the results of the global sequence alignment are in percent identity.
• the results, in percent identity must be manually corrected because the FASTDB program does not account for N- and C-terminal truncations of the interrogated sequence when calculating global percent identity.
• the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C- terminal of the interrogated sequence, that are not matched/aligned with a corresponding interrogated sequence residue, as a percent of the total bases of the query sequence.
• Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of the present invention. Only residues to the N- and C-termini of the interrogated sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query amino acid residues outside the farthest N- and C-terminal residues of the interrogated sequence.
• a 90 amino acid residue interrogated sequence is aligned with a 100- residue query sequence to determine percent identity.
• the deletion occurs at the N-terminus of the interrogated sequence and therefore, the FASTDB alignment does not match/align with the first residues at the N-terminus.
• the 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C- termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched, the final percent identity would be 90%.
• a 90-residue interrogated sequence is compared with a 100-residue query sequence. This time the deletions are internal so there are no residues at the N- or C- termini of the interrogated sequence, which are not matched/aligned with the query. In this case, the percent identity calculated by FASTDB is not manually corrected. Once again, only residue positions outside the N-and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequence are manually corrected. No other corrections are made for the purposes of the present invention.
• a polypeptide of interest is a fusion protein, and may contain, for example, an affinity tag domain or a reporter domain.
• Suitable affinity tags include any amino acid sequence that may be specifically bound to another moiety, usually another polypeptide, most usually an antibody.
• Suitable affinity tags include epitope tags, for example, the V5 tag, the FLAG tag, the HA tag (from hemagglutinin influenza virus), the myc tag, and the like, as is known in the art.
• Suitable affinity tags also include domains for which, binding substrates are known, e.g., HIS, GST and MBP tags, as is known in the art, and domains from other proteins for which specific binding partners, e.g., antibodies, particularly monoclonal antibodies, are available.
• Suitable affinity tags also include any protein-protein interaction domain, such as a IgG Fc region, which may be specifically bound and detected using a suitable binding partner, e.g. the IgG Fc receptor. It is expressly contemplated that such a fusion protein may contain a heterologous N-terminal domain (e.g., an epitope tag) fused in-frame with a GPCR that has had its N-terminal methionine residue either deleted or substituted with an alternative amino acid.
• a heterologous N-terminal domain e.g., an epitope tag
• Suitable reporter domains include any domain that can report the presence of a polypeptide. While it is recognized that an affinity tag may be used to report the presence of a polypeptide using, e.g., a labeled antibody that specifically binds to the tag, light emitting reporter domains are more usually used. Suitable light emitting reporter domains include luciferase (from, e.g., firefly, Vargula, Renilla reniformis or Renilla muelleri), or light emitting variants thereof.
• reporter domains include fluorescent proteins, (from e.g., jellyfish, corals and other coelenterates as such those from Aequoria, Renilla, Ptilosarcus, Stylatula species), or light emitting variants thereof.
• Light emitting variants of these reporter proteins are very well known in the art and may be brighter, dimmer, or have different excitation and/or emission spectra, as compared to a native reporter protein. For example, some variants are altered such that they no longer appear green, and may appear blue, cyan, yellow, enhanced yellow red (termed BFP, CFP, YFP eYFP and RFP, respectively) or have other emission spectra, as is known in the art.
• reporter domains include domains that can report the presence of a polypeptide through a biochemical or color change, such as ⁇ -galactosidase, ⁇ - glucuronidase, chloramphenicol acetyl transferase, and secreted embryonic alkaline phosphatase.
• an affinity tags or a reporter domain may be present at any position in a polypeptide of interest. However, in most embodiments, they are present at the C- or N-terminal end of a polypeptide of interest.
• GPCR coding sequences may be isolated from a library of
• GPCR coding sequence using any one or a combination of a variety of recombinant methods that do not need to be described herein. Subsequent substitution, deletion, and/or addition of nucleotides in the nucleic acid sequence encoding a protein may also be done using standard recombinant DNA techniques.
• site directed mutagenesis and subcloning may be used to
• nucleic acids encoding a polypeptide of interest may also be made by chemical synthesis entirely from oligonucleotides (e.g., Cello et al., Science (2002) 297: 1016-8).
• the codons of the nucleic acids encoding polypeptides of interest are optimized for expression in cells of a particular species, particularly a mammalian, e.g., mouse, rat, hamster, non-human primate, or human, species.
• the codons of the nucleic acids encoding polypeptides of interest are optimized for expression in cells of a particular species, particularly an amphibian species.
• the invention further provides vectors (also referred to as "constructs") comprising a subject nucleic acid.
• the subject nucleic acid sequences will be expressed in a host after the sequences have been operably linked to an expression control sequence, including, e.g. a promoter.
• the subject nucleic acids are also typically placed in an expression vector that can replicate in a host cell either as an episome or as an integral part of the host chromosomal DNA.
• expression vectors will contain selection markers, e.g., tetracycline or neomycin, to permit detection of those cells transformed with the desired DNA sequences (see, e.g., U.S. Pat. No. 4,704,362, which is incorporated herein by reference).
• Vectors, including single and dual expression cassette vectors are well known in the art
• Suitable vectors include viral vectors, plasmids, cosmids, artificial chromosomes (human artificial chromosomes, bacterial artificial chromosomes, yeast artificial chromosomes, etc.), mini-chromosomes, and the like. Retroviral, adenoviral and adeno-associated viral vectors may be used.
• expression vectors are available to those in the art for purposes of producing a polypeptide of interest in a cell and include expression vectors which are commercially available (e.g., from Invitrogen, Carlsbad, CA; Clontech, Mountain View, CA; Stratagene, La Jolla, CA).
• Commercially available expression vectors include, by way of non- limiting example, CMV promoter-based vectors.
• One suitable expression vector is pCMV.
• the expression vector may be adenoviral.
• An exemplary adenoviral vector may be purchased as AdEasyTM from Qbiogene (Carlsbad, CA) (He TC et al, Proc Natl Acad Sci USA (1998) 95:2509-2514; and US Patent No. 5,922,576).
• AdEasyTM from Qbiogene (Carlsbad, CA) (He TC et al, Proc Natl Acad Sci USA (1998) 95:2509-2514; and US Patent No. 5,922,
• the subject nucleic acids usually comprise an single open reading frame encoding a subject polypeptide of interest, however, in certain embodiments, since the host cell for expression of the polypeptide of interest may be a eukaryotic cell, e.g., a mammalian cell, such as a human cell, the open reading frame may be interrupted by introns.
• Subject nucleic acid are typically part of a transcriptional unit which may contain, in addition to the subject nucleic acid 3' and 5' untranslated regions (UTRs) which may direct RNA stability, translational efficiency, etc.
• the subject nucleic acid may also be part of an expression cassette which contains, in addition to the subject nucleic acid a promoter, which directs the transcription and expression of a polypeptide of interest, and a transcriptional terminator.
• Eukaryotic promoters can be any promoter that is functional in a eukaryotic host cell, including viral promoters and promoters derived from eukaryotic genes.
• Exemplary eukaryotic promoters include, but are not limited to, the following: the promoter of the mouse
• metallothionein I gene sequence (Hamer et al., J. Mol. Appl. Gen. 1:273-288, 1982); the TK promoter of Herpes virus (McKnight, Cell 31:355-365, 1982); the SV40 early promoter (Benoist et al., Nature (London) 290:304-310, 1981); the yeast gall gene sequence promoter (Johnston et al., Proc. Natl. Acad. Sci. (USA) 79:6971-6975, 1982); Silver et al., Proc. Natl. Acad. Sci.
• a promoter is used that is a promoter of the target pathogen. Promoters for use in the present invention are selected such that they are functional in the cell type (and/or animal) into which they are being introduced. In certain embodiments, the promoter is a CMV promoter.
• a subject vector may also provide for expression of a selectable marker.
• Suitable vectors and selectable markers are well known in the art and discussed in Ausubel, et al, (Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995) and Sambrook, et al, (Molecular Cloning: A Laboratory Manual, Third Edition, (2001) Cold Spring Harbor, N.Y.).
• a variety of different genes have been employed as selectable markers, and the particular gene employed in the subject vectors as a selectable marker is chosen primarily as a matter of convenience.
• selectable marker genes include: the thymidine kinase gene, the dihydrofolate reductase gene, the xanthine-guanine phosphoribosyl transferase gene, CAD, the adenosine deaminase gene, the asparagine synthetase gene, the antibiotic resistance genes, e.g. tetr, ampr, Cmr or cat, kanr or neor (aminoglycoside phosphotransferase genes), the hygromycin B phosphotransferase gene, and the like.
• polypeptides of interest may be fusion proteins that contain an affinity domain and/or a reporter domain.
• Methods for making fusions between a reporter or tag and a GPCR, for example, at the C- or N-terminus of the GPCR, are well within the skill of one of skill in the art (e.g. McLean et al, Mol. Pharma. Mol Pharmacol. 1999 56: 1182-91 ; Ramsay et al., Br. J. Pharmacology, 2001, 315-323) and will not be described any further.
• such a fusion protein may contain a heterologous N-terminal domain (e.g., an epitope tag) fused in-frame with a GPCR that has had its N-terminal methionine residue either deleted or substituted with an alternative amino acid.
• a polypeptide of interest may first be made from a native polypeptide and then operably linked to a suitable reporter/tag as described above.
• the subject nucleic acids may also contain restriction sites, multiple cloning sites, primer binding sites, ligatable ends, recombination sites etc., usually in order to facilitate the construction of a nucleic acid encoding a polypeptide of interest.
• the invention further provides host cells comprising a vector comprising a subject nucleic acid.
• Suitable host cells include prokaryotic, e.g., bacterial cells (for example E. coli), as well as eukaryotic cells e.g. an animal cell (for example an insect, mammal, fish, amphibian, bird or reptile cell), a plant cell (for example a maize or Arabidopsis cell), or a fungal cell (for example a S. cerevisiae cell).
• prokaryotic e.g., bacterial cells (for example E. coli), as well as eukaryotic cells e.g. an animal cell (for example an insect, mammal, fish, amphibian, bird or reptile cell), a plant cell (for example a maize or Arabidopsis cell), or a fungal cell (for example a S. cerevisiae cell).
• any cell suitable for expression of a polypeptide of interest-encoding nucleic acid may be used
• an animal host cell line is used, examples of which are as follows: monkey kidney cells (COS cells), monkey kidney CVI cells transformed by SV40 (COS-7, ATCC CRL 165 1); human embryonic kidney cells (HEK-293 ["293”], Graham et al. J. Gen Virol. 36:59 (1977)); HEK-293T ["293T”] cells; baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary-cells (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. (USA) 77:4216, (1980); Syrian golden hamster cells
• MCB3901 ATCC CRL-9595; mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CVI ATCC CCL 70); african green monkey kidney cells (VERO- 76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3 A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL 51); TRI cells (Mather et al., Annals N. Y. Acad. Sci 383:44-68 (1982)); NIH/3T3 cells (ATCC CRL-1658); and mouse L cells (ATCC CCL-1).
• melanophores are used.
• Melanophores are skin cells found in lower vertebrates. Relevant materials and methods will be followed according to the disclosure of U.S. Patent Number 5,462,856 and U.S. Patent Number 6,051,386.
• G protein receptor When a G protein receptor becomes active, it binds to a G protein (e.g., Gq, Gs, Gi, Gz, Go) and stimulates the binding of GTP to the G protein. The G protein then acts as a GTPase and slowly hydrolyzes the GTP to GDP, whereby the receptor, under normal conditions, becomes deactivated. However, activated receptors continue to exchange GDP to GTP.
• GTP GTPase
• GTPase e.g., Gq, Gs, Gi, Gz, Go
• candidate compounds are identified using the "generic" G protein-coupled receptor assay (i.e., an assay to select compounds that are agonists or inverse agonists), in some embodiments further screening to confirm that the compounds have interacted at the receptor site is preferred.
• a compound identified by the "generic” assay may not bind to the receptor, but may instead merely "uncouple" the G protein from the intracellular domain.
• Gs stimulates the enzyme adenylyl cyclase.
• Gi and Gz and Go
• Adenylyl cyclase catalyzes the conversion of ATP to cAMP; thus, activated GPCRs that couple the Gs protein are associated with increased cellular levels of cAMP.
• activated GPCRs that couple Gi (or Gz, Go) protein are associated with decreased cellular levels of cAMP. See, generally, "Indirect Mechanisms of Synaptic Transmission," Chpt. 8, From Neuron To Brain (3 ri Ed.) Nichols, J.G. et al eds. Sinauer Associates, Inc. (1992).
• assays that detect cAMP can be utilized to determine if a candidate compound is, e.g., an inverse agonist to the receptor (i.e., such a compound would decrease the levels of cAMP).
• a candidate compound e.g., an inverse agonist to the receptor
• a variety of approaches known in the art for measuring cAMP can be utilized; in some embodiments a preferred approach relies upon the use of anti-cAMP antibodies in an ELISA-based format.
• Another type of assay that can be utilized is a whole cell second messenger reporter system assay. Promoters on genes drive the expression of the proteins that a particular gene encodes.
• Cyclic AMP drives gene expression by promoting the binding of a cAMP-responsive DNA binding protein or transcription factor (CREB) that then binds to the promoter at specific sites called cAMP response elements and drives the expression of the gene.
• Reporter systems can be constructed which have a promoter containing multiple cAMP response elements before the reporter gene, e.g., ⁇ -galactosidase or luciferase.
• ⁇ -galactosidase or luciferase an activated Gs-linked receptor causes the accumulation of cAMP that then activates the gene and expression of the reporter protein.
• the reporter protein such as ⁇ -galactosidase or luciferase can then be detected using standard biochemical assays (Chen et al. 1995).
• Gq and Go are associated with activation of the enzyme phospholipase C, which in turn hydrolyzes the phospholipid PIP 2 , releasing two intracellular messengers: diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP 3 ). Increased accumulation of IP 3 is associated with activation of Gq- and Go-associated receptors. See, generally, “Indirect Mechanisms of Synaptic Transmission,” Chpt. 8, From Neuron To Brain (3 Ed.) Nichols, J.G. et al eds. Sinauer Associates, Inc. (1992).
• DAG diacylglycerol
• IP 3 inositol 1,4,5-triphosphate
• Assays that detect IP 3 accumulation can be utilized to determine if a candidate compound is, e.g., an inverse agonist to a Gq- or Go-associated receptor (i.e., such a compound would decrease the levels of IP 3 ).
• Gq-associated receptors can also been examined using an API reporter assay in that Gq-dependent phospholipase C causes activation of genes containing API elements; thus, activated Gq-associated receptors will evidence an increase in the expression of such genes, whereby inverse agonists thereto will evidence a decrease in such expression, and agonists will evidence an increase in such expression.
• Commercially available assays for such detection are available.
• an endogenous, constitutively active GPCR or a non-endogenous, constitutively activated GPCR for use in screening of candidate compounds for the direct identification of inverse agonists or agonists provides an interesting screening challenge in that, by definition, the receptor is active even in the absence of an endogenous ligand bound thereto.
• the receptor is active even in the absence of an endogenous ligand bound thereto.
• an approach be utilized that can enhance such differentiation.
• a preferred approach is the use of a GPCR Fusion Protein.
• a non-endogenous GPCR has been constitutively activated using the assay techniques set forth above (as well as others known to the art-skilled)
• screening take place using a mammalian or a melanophore expression system, as such a system will be expected to have endogenous G protein therein.
• the non-endogenous, constitutively activated GPCR will continuously signal.
• this signal be enhanced such that in the presence of, e.g., an inverse agonist to the receptor, it is more likely that it will be able to more readily differentiate, particularly in the context of screening, between the receptor when it is contacted with the inverse agonist.
• the GPCR Fusion Protein is intended to enhance the efficacy of G protein coupling with the GPCR.
• the GPCR Fusion Protein may be preferred for screening with either an endogenous, constitutively active GPCR or a non-endogenous, constitutively activated GPCR because such an approach increases the signal that is generated in such screening techniques. This is important in facilitating a significant "signal to noise" ratio; such a significant ratio is preferred for the screening of candidate compounds as disclosed herein.
• the construction of a construct useful for expression of a GPCR Fusion Protein is within the purview of those having ordinary skill in the art. Commercially available expression vectors and systems offer a variety of approaches that can fit the particular needs of an investigator.
• GPCR Fusion Protein construct Important criteria in the construction of such a GPCR Fusion Protein construct include but are not limited to, that the GPCR sequence and the G protein sequence both be in- frame (preferably, the sequence for the endogenous GPCR is upstream of the G protein sequence), and that the "stop" codon of the GPCR be deleted or replaced such that upon expression of the GPCR, the G protein can also be expressed.
• the GPCR can be linked directly to the G protein, or there can be spacer residues between the two (preferably, no more than about 12, although this number can be readily ascertained by one of ordinary skill in the art). Based upon convenience, it is preferred to use a spacer. In some embodiments, it is preferred that the G protein that couples to the non-endogenous GPCR will have been identified prior to the creation of the GPCR Fusion Protein construct.
• activated GPCRs that couple to Gi, Gz and Go are expected to inhibit the formation of cAMP making assays based upon these types of GPCRs challenging (i.e., the cAMP signal decreases upon activation, thus making the direct identification of, e.g., agonists (which would further decrease this signal) challenging).
• the cAMP signal decreases upon activation, thus making the direct identification of, e.g., agonists (which would further decrease this signal) challenging.
• it has been ascertained that for these types of receptors it is possible to create a GPCR Fusion Protein that is not based upon the GPCR's endogenous G protein, in an effort to establish a viable cyclase- based assay.
• an endogenous Gi coupled receptor can be fused to a Gs protein -such a fusion construct, upon expression, "drives” or “forces” the endogenous GPCR to couple with, e.g., Gs rather than the "natural" Gi protein, such that a cyclase-based assay can be established.
• a GPCR Fusion Protein is used and the assay is based upon detection of adenylyl cyclase activity, that the fusion construct be established with Gs (or an equivalent G protein that stimulates the formation of the enzyme adenylyl cyclase).
• G Protein Fusion construct that utilizes a Gq Protein fused with a Gs, Gi, Gz or Go Protein.
• a preferred fusion construct can be accomplished with a Gq Protein wherein the first six (6) amino acids of the G-protein a-subunit ("Gaq") is deleted and the last five (5) amino acids at the C-terminal end of Gaq is replaced with the corresponding amino acids of the Ga of the G protein of interest.
• a fusion construct can have a Gq (6 amino acid deletion) fused with a Gi Protein, resulting in a "Gq/Gi Fusion Construct".
• This fusion construct will force the endogenous Gi coupled receptor to couple to its non-endogenous G protein, Gq, such that the second messenger, for example, inositol triphosphate or diacylglycerol, can be measured in lieu of cAMP production.
• Gq non-endogenous G protein
• a Gi coupled receptor is known to inhibit adenylyl cyclase, and, therefore, decreases the level of cAMP production, which can make the assessment of cAMP levels challenging.
• an effective technique in measuring the decrease in production of cAMP as an indication of activation of a receptor that predominantly couples Gi upon activation can be accomplished by co-transfecting a signal enhancer, e.g., a non-endogenous, constitutively activated receptor that predominantly couples with Gs upon activation (e.g., TSHR-A623I; see infra), with the Gi linked GPCR.
• a signal enhancer e.g., a non-endogenous, constitutively activated receptor that predominantly couples with Gs upon activation (e.g., TSHR-A623I; see infra)
• activation of a Gs coupled receptor can be determined based upon an increase in production of cAMP.
• Activation of a Gi coupled receptor leads to a decrease in production cAMP.
• the co-transfection approach is intended to advantageously exploit these "opposite” affects.
• co-transfection of a non- endogenous, constitutively activated Gs coupled receptor (the "signal enhancer") with expression vector alone provides a baseline cAMP signal (i.e., although the Gi coupled receptor will decrease cAMP levels, this "decrease” will be relative to the substantial increase in cAMP levels established by constitutively activated Gs coupled signal enhancer).
• the signal enhancer By then co- transfecting the signal enhancer with the "target receptor", an inverse agonist of the Gi coupled target receptor will increase the measured cAMP signal, while an agonist of the Gi coupled target receptor will decrease this signal.
• Candidate compounds that are directly identified using this approach should be assessed independently to ensure that these do not target the signal enhancing receptor (this can be done prior to or after screening against the co-transfected receptors).
• IC50 value is determined using an assay selected from the group consisting of: IP3 assay carried out using transfected HEK293 cells expressing recombinant H3R polypeptide; and melanophore assay carried out using transfected melanophores expressing recombinant H3R polypeptide.
• the test compound is an inverse agonist or antagonist with an IC50 of less than 10 ⁇ , of less than 1 ⁇ , of less than 100 nM, or of less than 10 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 10 ⁇ in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 9 ⁇ in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 8 ⁇ in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 7 ⁇ in the assay.
• the test compound is an inverse agonist or antagonist with an IC50 of less than 6 ⁇ in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 5 ⁇ in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 4 ⁇ in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 3 ⁇ in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 2 ⁇ in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 1 ⁇ in the assay.
• the test compound is an inverse agonist or antagonist with an IC50 of less than 900 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 800 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 700 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 600 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 500 nM in the assay.
• the test compound is an inverse agonist or antagonist with an IC50 of less than 400 nM n the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 300 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 200 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 100 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 90 nM in the assay.
• the test compound is an inverse agonist or antagonist with an IC50 of less than 80 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 70 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 60 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 50 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 40 nM n the assay.
• the test compound is an inverse agonist or antagonist with an IC50 of less than 30 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 20 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 10 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 in the assay of a value selected from the interval of 1 nM to 10 ⁇ . In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 in the assay of a value selected from the interval of 1 nM to 1 ⁇ .
• the test compound is an inverse agonist or antagonist with an IC50 in the assay of a value selected from the interval of 1 nM to 100 nM. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 in the assay of a value selected from the interval of 1 nM to 10 nM. In some embodiments, the test compound is selective for the GPCR.
• Melanophores are skin cells found in lower vertebrates. They contain pigmented organelles termed melanosomes. Melanophores are able to redistribute these melanosomes along a microtubule network upon G-protein coupled receptor (GPCR) activation. The result of this pigment movement is an apparent lightening or darkening of the cells.
• GPCR G-protein coupled receptor
• melanophores the decreased levels of intracellular cAMP that result from activation of a Gi-coupled receptor cause melanosomes to migrate to the center of the cell, resulting in a dramatic lightening in color. If cAMP levels are then raised, following activation of a Gs-coupled receptor, the melanosomes are re-dispersed and the cells appear dark again.
• the increased levels of diacylglycerol that result from activation of Gq-coupled receptors can also induce this re-dispersion.
• the technology is also suited to the study of certain receptor tyrosine kinases.
• the response of the melanophores takes place within minutes of receptor activation and results in a simple, robust color change. The response can be easily detected using a conventional absorbance microplate reader or a modest video imaging system. Unlike other skin cells, the melanophores derive from the neural crest and appear to express a full complement of signaling proteins. In particular, the cells express an extremely wide range of G-proteins and so are able to functionally express almost all GPCRs.
• Melanophores can be utilized to identify compounds, including natural ligands, which bind to and/or activate GPCRs.
• This method can be conducted by introducing test cells of a pigment cell line capable of dispersing or aggregating their pigment in response to a specific stimulus and expressing an exogenous clone coding for the GPCR.
• An initial state of pigment disposition can be set using, for example, using melatonin, MSH or light.
• the test cells are then contacted with chemical compounds, and it is determined whether the pigment disposition in the cells changed from the initial state of pigment disposition. Dispersion of pigments cells due to the candidate compound, including but not limited to a ligand, coupling to the GPCR will appear dark on a petri dish, while aggregation of pigments cells will appear light.
• Melanophores are transfected by electroporation with a plasmid which contains the coding sequence of mouse or human H3R.
• the cells are plated in 96-well plates. 48 hours post- transfection, half of the cells on each plate are treated with lOnM melatonin.
• Melatonin activates an endogenous Gi-coupled receptor in the melanophores and causes them to aggregate their pigment.
• the remaining half of the cells are transferred to serum-free medium 0.7X L-15 (Gibco). After one hour, the cells in serum- free media remain in a pigment-dispersed state while the melatonin-treated cells are in a pigment-aggregated state.
• the cells are treated with different compounds from a proprietary compound library containing 140,000-150,000 organic small molecule compounds. If H3R bound to the compound, the melanophores would be expected to undergo a color change, for example, due to pigment aggregation, in response to the compound.
• a G protein-coupled receptor When a G protein-coupled receptor is in its active state, either as a result of ligand binding or constitutive activation, the receptor couples to a G protein and stimulates the release of GDP and subsequent binding of GTP to the G protein.
• the alpha subunit of the G protein-receptor complex acts as a GTPase and slowly hydrolyzes the GTP to GDP, at which point the receptor normally is deactivated. Activated receptors continue to exchange GDP for GTP.
• the non-hydrolyzable GTP analog, [ 35 S]GTPyS can be utilized to demonstrate enhanced binding of [ 35 S]GTPyS to membranes expressing activated receptors.
• the assay utilizes the ability of G protein coupled receptors to stimulate [ 35 S]GTPyS binding to membranes expressing the relevant receptors.
• the assay can, therefore, be used in the direct identification method to screen candidate compounds to endogenous GPCRs and non- endogenous, constitutively activated GPCRs.
• the assay is generic and has application to drug discovery at all G protein-coupled receptors.
• the [ 35 S]GTPyS assay is incubated in 20 mM HEPES and between 1 and about 20mM MgCl 2 (this amount can be adjusted for optimization of results, although 20mM is preferred) pH 7.4, binding buffer with between about 0.3 and about 1.2 nM [ 35 S]GTPyS (this amount can be adjusted for optimization of results, although 1.2 is preferred) and 12.5 to 75 ⁇ g membrane protein (e.g, 293 cells expressing the H3R; this amount can be adjusted for optimization) and 10 ⁇ GDP (this amount can be changed for optimization) for 1 hour. Wheatgerm agglutinin beads (25 ⁇ ; Amersham) are then added and the mixture incubated for another 30 minutes at room temperature. The tubes are then centrifuged at 1500 x g for 5 minutes at room temperature and then counted in a scintillation counter.
• a Flash PlateTM Adenylyl Cyclase kit (New England Nuclear; Cat. No. SMP004A) designed for cell-based assays can be modified for use with crude plasma membranes.
• the Flash Plate wells can contain a scintillant coating which also contains a specific antibody recognizing cAMP.
• the cAMP generated in the wells can be quantitated by a direct competition for binding of radioactive cAMP tracer to the cAMP antibody. The following serves as a brief protocol for the measurement of changes in cAMP levels in whole cells that express a receptor.
• Transfected cells are harvested approximately twenty four hours after transient transfection. Media is carefully aspirated off and discarded. 10 mL of PBS is gently added to each dish of cells followed by careful aspiration. 1 mL of Sigma cell dissociation buffer and 3 mL of PBS are added to each plate. Cells are pipetted off the plate and the cell suspension is collected into a 50 mL conical centrifuge tube. Cells are then centrifuged at room temperature at 1,100 rpm for 5 minutes. The cell pellet is carefully re-suspended into an appropriate volume of PBS (about 3 mL/plate). The cells are then counted using a hemocytometer and additional PBS is added to give the appropriate number of cells (with a final volume of about 50 ⁇ ).
• cAMP standards and Detection Buffer comprising ⁇ of tracer [ 125 ⁇ ] cAMP (50 ⁇ ) to 11 mL Detection Buffer
• Assay Buffer is prepared fresh for screening and contains 50 ⁇ L ⁇ of Stimulation Buffer, 3 ⁇ L ⁇ of candidate compound (12 ⁇ final assay concentration) and 50 ⁇ L ⁇ cells.
• Assay Buffer is stored on ice until utilized.
• the assay preferably carried out, for example, in a 96-well plate, is initiated by addition of 50 ⁇ L ⁇ of cAMP standards to appropriate wells followed by addition of 50 ⁇ , of PBSA to wells HI 1 and H12.
• Stimulation Buffer 50 ⁇ , of Stimulation Buffer is added to all wells.
• DMSO or selected candidate compounds
• DMSO or selected candidate compounds
• the cells are then added to the wells and incubated for 60 minutes at room temperature.
• 100 ⁇ L ⁇ of Detection Mix containing tracer cAMP is then added to the wells. Plates are then incubated additional 2 hours followed by counting in a Wallac MicroBeta scintillation counter. Values of cAMP/well are then extrapolated from a standard cAMP curve which is contained within each assay plate.
• TSHR is a Gs coupled GPCR that causes the accumulation of cAMP upon activation.
• TSHR can be constitutively activated by mutating amino acid residue 623 (i.e., changing an alanine residue to an isoleucine residue).
• a Gi coupled receptor is expected to inhibit adenylyl cyclase, and, therefore, decrease the level of cAMP production, which can make assessment of cAMP levels challenging.
• An effective technique for measuring the decrease in production of cAMP as an indication of activation of a Gi coupled receptor can be accomplished by co-transfecting, non- endogenous, constitutively activated TSHR (TSHR-A623I) (or an endogenous, constitutively active Gs coupled receptor) as a "signal enhancer" with a Gi linked target GPCR to establish a baseline level of cAMP.
• TSHR-A623I non- endogenous, constitutively activated TSHR
• Gs coupled receptor an endogenous, constitutively active Gs coupled receptor
• the target GPCR is then co-transfected with the signal enhancer, and it is this material that can be used for screening.
• this approach is preferably used in the direct identification of candidate compounds against Gi coupled receptors. It is noted that for a Gi coupled GPCR, when this approach is used, an inverse agonist of the target GPCR will increase the cAMP signal and an agonist will decrease the cAMP signal.
• tube A is prepared by mixing 2 ⁇ g DNA of each receptor transfected into the mammalian cells, for a total of 4 ⁇ g DNA (e.g., pCMV vector; pCMV vector with mutated THSR (TSHR-A623I); TSHR-A623I and GPCR, etc.) in 1.2 mL serum free DMEM (Irvine Scientific, Irvine, CA); tube B is prepared by mixing 120 ⁇ L ⁇ lipofectamine (Gibco BRL) in 1.2 mL serum free DMEM.
• Tubes A and B are then admixed by inversions (several times), followed by incubation at room temperature for 30-45minutes.
• the admixture is referred to as the "transfection mixture”.
• Plated 293 cells are washed with 1XPBS, followed by addition of 10 mL serum free DMEM.
• 2.4 mL of the transfection mixture is then added to the cells, followed by incubation for 4 hours at 37°C/5% C0 2 .
• the transfection mixture is then removed by aspiration, followed by the addition of 25 mL of DMEM/10% Fetal Bovine Serum. Cells are then incubated at 37°C/5% C0 2 . After 24 hours incubation, cells are harvested and utilized for analysis.
• a Flash PlateTM Adenylyl Cyclase kit (New England Nuclear; Cat. No. SMP004A) is designed for cell-based assays, but can be modified for use with crude plasma membranes depending on the need of the skilled artisan.
• the Flash Plate wells contain a scintillant coating which also contains a specific antibody recognizing cAMP.
• the cAMP generated in the wells can be quantitated by a direct competition for binding of radioactive cAMP tracer to the cAMP antibody. The following serves as a brief protocol for the measurement of changes in cAMP levels in whole cells that express a receptor of interest.
• Transfected cells are harvested approximately twenty four hours after transient transfection. Media is carefully aspirated off and discarded. 10 mL of PBS is gently added to each dish of cells followed by careful aspiration. 1 mL of Sigma cell dissociation buffer and 3 mL of PBS is added to each plate. Cells are pipetted off the plate and the cell suspension is collected into a 50 mL conical centrifuge tube. Cells are then centrifuged at room temperature at 1,100 rpm for 5 minutes. The cell pellet is carefully re-suspended into an appropriate volume of PBS (about 3 mL/plate). The cells are then counted using a hemocytometer and additional PBS is added to give the appropriate number of cells (with a final volume of about 50 ⁇ /well).
• cAMP standards and Detection Buffer comprising ⁇ of tracer [ 125 ⁇ ] cAMP (50 ⁇ ) to 11 mL Detection Buffer
• Assay Buffer should be prepared fresh for screening and contain 50 ⁇ L ⁇ of Stimulation Buffer, 3 ⁇ L ⁇ of candidate compound (12 ⁇ final assay concentration) and 50 ⁇ L ⁇ cells.
• Assay Buffer can be stored on ice until utilized. The assay can be initiated by addition of 50 ⁇ L ⁇ of cAMP standards to appropriate wells followed by addition of 50 ⁇ L ⁇ of PBS A to wells H-l 1 and H12. Fifty ⁇ L ⁇ o ⁇ Stimulation Buffer is added to all wells.
• Selected compounds e.g., TSH
• TSH Selected compounds
• the cells are then added to the wells and incubated for 60 minutes at room temperature.
• 100 ⁇ L ⁇ of Detection Mix containing tracer cAMP is then added to the wells. Plates are then incubated additional 2 hours followed by counting in a Wallac MicroBeta scintillation counter. Values of cAMP/well are extrapolated from a standard cAMP curve which is contained within each assay plate.
• 293 or 293T cells are plated-out on 96 well plates at a density of 2 x 10 4 cells per well and are transfected using Lipofectamine Reagent (BRL) the following day according to manufacturer instructions.
• BBL Lipofectamine Reagent
• a DNA lipid mixture is prepared for each 6-well transfection as follows: 260ng of plasmid DNA in 100 ⁇ .
• DMEM DMEM lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing 2 lipid.
• the 260ng of plasmid DNA consists of 200ng of a 8xCRE-Luc reporter plasmid, 50ng of pCMV comprising endogenous receptor or non-endogenous receptor or pCMV alone, and lOng of a GPRS expression plasmid (GPRS in pcDNA3 (Invitrogen)).
• the 8XCRE-Luc reporter plasmid is prepared as follows: vector SRIF- -gal is obtained by cloning the rat somatostatin promoter (
• Eight (8) copies of cAMP response element are obtained by PCR from an adenovirus template AdpCF126CCRE8 (see, Suzuki et al., Hum Gene Ther 7: 1883-1893 (1996)) and cloned into the SRIF- -gal vector at the Kpn-BglV site, resulting in the 8xCRE- -gal reporter vector.
• the 8xCRE-Luc reporter plasmid is generated by replacing the beta-galactosidase gene in the 8xCRE- -gal reporter vector with the luciferase gene obtained from the pGL3-basic vector (Promega) at the Hindlll-BamHI site.
• the DNA/lipid mixture is diluted with 400 ⁇ of DMEM and 100 ⁇ of the diluted mixture is added to each well.
• 100 ⁇ of DMEM with 10% FCS are added to each well after a four hour incubation in a cell culture incubator.
• the transfected cells are changed with 200 ⁇ /well of DMEM with 10% FCS.
• the wells are changed to 100 ⁇ /well of DMEM without phenol red, after one wash with PBS.
• Luciferase activity is measured the next day using the LucLite reporter gene assay kit (Packard) following manufacturer instructions and read on a 1450 MicroBetaTM scintillation and luminescence counter (Wallac).
• a method to detect Gq stimulation depends on the known property of Gq-dependent phospholipase C to cause the activation of genes containing API elements in their promoter.
• a PathdetectTM AP-1 cis-Reporting System (Stratagene, Catalogue No. 219073) can be utilized following the protocol set forth above with respect to the CREB reporter assay, except that the components of the calcium phosphate precipitate are 410 ng pAPl-Luc, 80 ng pCMV-receptor expression plasmid, and 20 ng CMV-SEAP.
• Gq-dependent phospholipase C One method to detect Gq stimulation depends on the known property of Gq-dependent phospholipase C to cause the activation of genes containing serum response factors in their promoter.
• a PathdetectTM SRF-Luc-Reporting System (Stratagene) can be utilized to assay for Gq coupled activity in, for example, COS7 cells. Cells are transfected with the plasmid components of the system and the indicated expression plasmid encoding endogenous or non-endogenous GPCR using a Mammalian TransfectionTM Kit (Stratagene, Catalogue #200285) according to the manufacturer's instructions.
• 410 ng SRF-Luc, 80 ng pCMV-receptor expression plasmid and 20 ng CMV-SEAP secreted alkaline phosphatase expression plasmid; alkaline phosphatase activity is measured in the media of transfected cells to control for variations in transfection efficiency between samples
• CMV-SEAP secreted alkaline phosphatase expression plasmid; alkaline phosphatase activity is measured in the media of transfected cells to control for variations in transfection efficiency between samples
• Cells are then lysed and assayed for luciferase activity using a LucliteTM Kit (Packard, Cat. No. 6016911) and "Trilux 1450 Microbeta" liquid scintillation and luminescence counter (Wallac) as per the manufacturer's instructions.
• the data can be analyzed using GraphPad PrismTM 2.0a (GraphPad Software Inc.).
• cells comprising the receptor of interest can be plated onto 24 well plates, usually lxlO 5 cells/well (although this number can be optimized).
• cells can be transfected by first mixing 0.25 ⁇ g DNA in 50 ⁇ serum free DMEM/well and 2 ⁇ lipofectamine in 50 ⁇ serum free DMEM/well. The solutions are gently mixed and incubated for 15-30 minutes at room temperature. Cells are washed with 0.5 mL PBS and 400 ⁇ of serum free media is mixed with the transfection media and added to the cells. The cells are then incubated for 3-4 hours at 37°C/5%C0 2 and then the transfection media is removed and replaced with 1 mL/well of regular growth media.
• the cells are labeled with 3 H-myo-inositol. Briefly, the media is removed and the cells are washed with 0.5 mL PBS. Then 0.5 mL inositol-free/serum free media (GIBCO BRL) is added/well with 0.25 ⁇ of 3 H-myo-inositol/ well and the cells are incubated for 16-18 hours overnight at 37°C/5%C0 2 .
• GEBCO BRL inositol-free/serum free media
• the cells are washed with 0.5 mL PBS and 0.45 mL of assay medium is added containing inositol-free/serum free media, 10 ⁇ pargyline, 10 mM lithium chloride or 0.4 mL of assay medium and 50 ⁇ L ⁇ of lOx ketanserin (ket) to final concentration of 10 ⁇ , if using a control construct containing a serotonin receptor.
• the cells are then incubated for 30 minutes at 37°C.
• the cells are then washed with 0.5 mL PBS and 200 ⁇ L ⁇ of fresh/ice cold stop solution (1M KOH; 18 mM Na-borate; 3.8 mM EDTA) is added/well.
• the solution is kept on ice for 5-10 minutes or until cells were lysed and then neutralized by 200 ⁇ L ⁇ of fresh/ice cold neutralization sol. (7.5 % HCL).
• the lysate is then transferred into 1.5 mL eppendorf tubes and 1 mL of chloroform/methanol (1:2) is added/tube.
• the solution is vortexed for 15 seconds and the upper phase is applied to a Biorad AG1-X8TM anion exchange resin (100-200 mesh). Firstly, the resin is washed with water at 1: 1.25 W/V and 0.9 mL of upper phase is loaded onto the column.
• the column is washed with 10 mL of 5 mM myo-inositol and 10 mL of 5 mM Na- borate/60mM Na-formate.
• the inositol tris phosphates are eluted into scintillation vials containing 10 mL of scintillation cocktail with 2 mL of 0.1 M formic acid/ 1 M ammonium formate.
• the columns are regenerated by washing with 10 mL of 0.1 M formic acid/3 M ammonium formate and rinsed twice with dd H 2 0 and stored at 4°C in water.
• membranes comprising the Target GPCR of interest for use in the identification of candidate compounds as, e.g.,. agonists, inverse agonists or antagonists are prepared as follows:
• “Membrane Scrape Buffer” is comprised of 20mM HEPES and lOmM EDTA, pH 7.4;
• “Membrane Wash Buffer” is comprised of 20mM HEPES and 0. lmM EDTA, pH 7.4;
• “Binding Buffer” is comprised of 20mM HEPES, 100 mM NaCl, and 10 mM MgCl 2 , pH 7.4.
• the media is aspirated from a confluent monolayer of cells, followed by rinsing with lOmL cold PBS, followed by aspiration. Thereafter, 5 mL of Membrane Scrape Buffer is added to scrape cells; this is followed by transfer of cellular extract into 50 mL centrifuge tubes (centrifuged at 20,000 rpm for 17 minutes at 4°C). Thereafter, the supernatant is aspirated and the pellet is resuspended in 30 mL Membrane Wash Buffer followed by centrifuge at 20,000 rpm for 17 minutes at 4°C. The supernatant is then aspirated and the pellet resuspended in Binding Buffer. This is then homogenized using a Brinkman PolytronTM homogenizer (15-20 second bursts until the all material is in suspension). This is referred to herein as "Membrane Protein".
• Protein concentration of the membranes is determined using the Bradford Protein Assay (protein can be diluted to about 1.5 mg/mL, aliquoted and frozen (-80°C) for later use; when frozen, protocol for use will be as follows: on the day of the assay, frozen Membrane Protein is thawed at room temperature, followed by vortex and then homogenized with a Polytron at about 12 x 1,000 rpm for about 5-10 seconds; it is noted that for multiple preparations, the homogenizer should be thoroughly cleaned between homogenization of different preparations).
• Binding Buffer (as per above); Bradford Dye Reagent; Bradford Protein Standard is utilized, following manufacturer instructions (Biorad, cat. no. 500-0006).
• Duplicate tubes are prepared, one including the membrane, and one as a control "blank". Each tube contains 800 ⁇ L ⁇ Binding Buffer. Thereafter, 10 ⁇ L ⁇ of Bradford Protein Standard (Img/mL) is added to each tube, and 10 ⁇ L ⁇ of membrane Protein is then added to just one tube (not the blank). Thereafter, 200 ⁇ L ⁇ of Bradford Dye Reagent is added to each tube, followed by vortexing of each tube. After five (5) minutes, the tubes are re-vortexed and the material therein is transferred to cuvettes. The cuvettes are read using a CECIL 3041 spectrophotometer, at wavelength 595.
• GDP Buffer consists of 37.5 mL Binding Buffer and 2 mg GDP (Sigma, cat. no. G-7127), followed by a series of dilutions in Binding Buffer to obtain 0.2 ⁇ GDP (final concentration of GDP in each well is 0.1 ⁇ GDP); each well comprising a candidate compound has a final volume of 200 consisting of 100 ⁇ GDP Buffer (final concentration, ⁇ . ⁇ GDP), 50 ⁇ Membrane Protein in Binding Buffer, and 50 ⁇ [ 35 S]GTPyS (0.6 nM) in Binding Buffer (2.5 ⁇ [ 35 S]GTPyS per 10 mL Binding Buffer).
• Candidate compounds can be screened using a 96-well plate format (these can be frozen at -80°C).
• Membrane Protein or membranes with expression vector excluding the Target GPCR, as control), are homogenized briefly until in suspension. Protein concentration is be determined using the Bradford Protein Assay set forth above. Membrane Protein (and control) is diluted to
• Binding Buffer 0.25mg/mL in Binding Buffer (final assay concentration, 12 ⁇ g/well). Thereafter, 100 ⁇ L ⁇ GDP Buffer is added to each well of a Wallac ScintistripTM (Wallac).
• a 5 ⁇ ⁇ pin-tool is used to transfer 5 ⁇ L ⁇ of a candidate compound into such well (i.e., 5 ⁇ L ⁇ in total assay volume of 200 ⁇ L ⁇ is a 1:40 ratio such that the final screening concentration of the candidate compound is 10 ⁇ ).
• the pin tool should be rinsed in three reservoirs comprising water (IX), ethanol (IX) and water (2X) - excess liquid should be shaken from the tool after each rinse and dried with paper and kimwipes.
• Another assay approach for identifying candidate compounds as, e.g., agonists, inverse agonist, or antagonists can accomplished by utilizing a cyclase-based assay.
• this assay approach can be utilized as an independent approach to provide confirmation of the results from the [ 35 S]GTPyS approach as set forth in the above example.
• SMP004A can be utilized for direct identification of candidate compounds as inverse agonists and agonists to a receptor of interest in accordance with the following protocol.
• Transfected cells are harvested approximately three days after transfection.
• Membranes are prepared by homogenization of suspended cells in buffer containing 20 mM HEPES, pH 7.4 and 10 mM MgCl 2 . Homogenization is performed on ice using a Brinkman PolytronTM for approximately 10 seconds. The resulting homogenate is centrifuged at 49,000 X g for 15 minutes at 4°C. The resulting pellet is then resuspended in buffer containing 20 mM HEPES, pH 7.4 and 0.1 mM EDTA, homogenized for 10 seconds, followed by centrifugation at 49,000 x g for 15 minutes at 4°C. The resulting pellet is then stored at -80°C until utilized.
• the membrane pellet On the day of direct identification screening, the membrane pellet is slowly thawed at room temperature, resuspended in buffer containing 20mM HEPES, pH 7.4 and lOmM MgCl 2 , to yield a final protein concentration of 0.60 mg/mL (the resuspended membranes are placed on ice until use).
• cAMP standards and Detection Buffer comprising 2 ⁇ of tracer [ 125 I]cAMP (100 ⁇ ) to 11 mL Detection Buffer] are prepared and maintained in accordance with the manufacturer' s instructions.
• Assay Buffer is prepared fresh for screening and contains 20mM HEPES, pH 7.4, 10 mM MgCl 2 , 20mM phospocreatine (Sigma), 0.1 units/mL creatine phosphokinase (Sigma), 50 ⁇ GTP (Sigma), and 0.2 mM ATP (Sigma); Assay Buffer is then stored on ice until utilized.
• Candidate compounds are added to, for example, 96-well plate wells (3 ⁇ /well; 12 ⁇ final assay concentration), together with 40 ⁇ Membrane Protein (30 ⁇ g/well) and 50 ⁇ of Assay Buffer. This admixture is then incubated for 30 minutes at room temperature, with gentle shaking.
• Target Receptor (experimental) and pCMV (negative control) stably transfected cells from respective clonal lines are seeded into poly-D-lysine pretreated 96-well plates (Becton-Dickinson, #356640) at 5.5xl0 4 cells/well with complete culture medium (DMEM with 10% FBS, 2 mM L- glutamine, lmM sodium pyruvate) for assay the next day.
• DMEM complete culture medium
• a promiscuous G protein such as Gal 5, Gal6, or the chimeric Gq/Gi alpha subunit is available to cause a detectable calcium flux.
• Fluo4-AM (Molecular Probe, #F14202) incubation buffer stock, 1 mg Fluo4-AM is dissolved in 467 ⁇ DMSO and 467 ⁇ Pluoronic acid (Molecular Probe, #P3000) to give a lmM stock solution that can be stored at -20°C for a month.
• Fluo4-AM is a fluorescent calcium indicator dye.
• Candidate compounds are prepared in wash buffer (IX HBSS/2.5mM Probenicid/20mM HEPES at pH 7.4).
• culture medium is removed from the wells and the cells are loaded with ⁇ of 4 ⁇ Fluo4-AM/2.5 mM Probenicid (Sigma, #P8761)/20mM HEPES/complete medium at pH 7.4. Incubation at 37°C/5% C0 2 is allowed to proceed for 60 minutes.
• the Fluo4-AM incubation buffer is removed and the cells are washed 2X with 100 ⁇ wash buffer. In each well is left 100 ⁇ wash buffer. The plate is returned to the incubator at 37°C/5% C0 2 for 60 minutes.
• FLIPR Fluorometric Imaging Plate Reader
• MAP kinase mitogen activated kinase
• MAP kinase can be detected by several approaches.
• One approach is based on an evaluation of the phosphorylation state, either unphosphorylated (inactive) or phosphorylated (active).
• the phosphorylated protein has a slower mobility in SDS-PAGE and can therefore be compared with the unstimulated protein using Western blotting.
• antibodies specific for the phosphorylated protein are available (New England Biolabs) which can be used to detect an increase in the phosphorylated kinase.
• cells are stimulated with the candidate compound and then extracted with Laemmli buffer. The soluble fraction is applied to an SDS- PAGE gel and proteins are transferred electrophoretically to nitrocellulose or Immobilin.
• Immunoreactive bands are detected by standard Western blotting technique. Visible or chemiluminescent signals are recorded on film and can be quantified by densitometry.
• Another approach is based on evalulation of the MAP kinase activity via a phosphorylation assay.
• Cells are stimulated with the candidate compound and a soluble extract is prepared.
• the extract is incubated at 30°C for 10 minutes with gamma- 32 P-ATP, an ATP regenerating system, and a specific substrate for MAP kinase such as phosphorylated heat and acid stable protein regulated by insulin, or PHAS-I.
• the reaction is terminated by the addition of H 3 P0 4 and samples are transferred to ice.
• An aliquot is spotted onto Whatman P81 chromatography paper, which retains the phosphorylated protein.
• the chromatography paper is washed and counted for 32 P is a liquid scintillation counter.
• the cell extract is incubated with gamma- 32 P-ATP, an ATP regenerating system, and biotinylated myelin basic proein bound by streptavidin to a filter support.
• the myelin basic protein is a substrate for activated MAP kinase.
• the phosphorylation reaction is carried out for 10 minutes at 30°C.
• the extract can then be aspirated through the filter, which retains, the phosphorylated myelin basic protein.
• the filter is washed and counted for 32 P by liquid scintillation counting.
• pruritus model that displays at least one element or aspect of pruritus and are amenable to the evaluation of anti-pruritic agents.
• the pruritus model is a behavioral model.
• the pruritus model is a mechanistic or molecular model.
• the pruritus model is a vertebrate model. In another embodiment, the pruritus model is a mammalian model. In another embodiment, the pruritus model is a primate model, a dog model, a guinea pig model, a mouse model, or a cat model.
• animal models may be particularly useful in analyzing potential anti-pruritic agents.
• animal pruritus models can be used to determine the in vivo efficacy of a H3R antagonist.
• the pruritus model is behaviorally based and evaluation of such a model comprises evaluating the behavior of the organism that has been administered a test compound.
• the pruritus model is selected from the group: pruritogen injection model, passive cutaneous anaphylaxis model, allergic pruritus model, and spontaneous pruritus model.
• the pruritus model is selected from the group: histamine induced pruritus model, DNP-Ovalbumin pruritus model, and DNFB pruritus model.
• the response to pruritus is a behavioral response.
• the following systems are described for purposes of illustration and are not intended to be limiting.
• itch is induced by an intradermal or subcutaneous injection of a pruritogen (i.e. histamine) into the rostral part of the back of mice. Mice are video recorded and the number of scratching of the injection site by a hind leg after pruritogen injection is counted.
• a pruritogen i.e. histamine
• Mice are video recorded and the number of scratching of the injection site by a hind leg after pruritogen injection is counted.
• Several substances in addition to histamine can be injected to induce itch: serotonin, substance P, chloroquine, protease-activated receptor 2 (PAR-2) activating peptide, trypsin.
• a test compound can be administered orally or by other routes shortly prior to itch induction, and the efficacy of the test compound compared to that of a sham treatment or that of a positive control drug.
• itch is triggered not by direct injection of a pruritogen but rather by an anaphylactic reaction upon injection of an antigen to antibody-primed mice.
• a small amount of IgE antibody directed against a known antigen i.e., dinitrophenyl-conjugated ovalbumin or DNP-OVA
• the antigen is later given to induce an allergic reaction, which results in itch.
• 20-100 ng of dinitrophenyl-IgE is injected into the rostral part of the back of the animals, and 24 hours laterlOO ug of antigen (DNP-OVA) is injected intraperitoneally.
• a test compound can be dosed 30 minutes before antigen injection.
• Allergic pruritus model differs from the passive cutaneous anaphylaxis model in that sensitization to an antigen develops through an active immunization process (i.e. hypersensitivity develops to a known antigen via an endogenously generated immune response).
• an antigen DNP-OVA is administered by intraperitoneal injection in mice.
• a booster immunization may be given a week later.
• Pruritus is then induced two weeks after the initial immunization by injecting the antigen at the rostral part of the back. Again, a test compound is dosed shortly before itch induction.
• antigen may be toluene-2,4-diisocyanate (TDI), 2,4-niditrochlorobenzene (DNCB), oxazolone, or picryl chloride.
• Sensitization can be done by injection intradermally, intraperitoneally, subcutaneously or by other routes.
• An antigen can be given by topical application, by intradermal or subcutaneous injection to induce an allergic reaction.
• a test compound can be administered before or during the sensitization period or shortly before the allergen challenge.
• mice develop pruritus due to ongoing inflammation without the need for pruritogen or antigen injection.
• spontaneous models include the NC/Nga mice, interleukin-4 and interleukin-13 transgenic mice, and MRL/lpr mice.
• NC/Nga mice develop skin lesions and pruritus after 8 weeks of age when kept in conventional vivarium conditions (Takano, N. et al., 2003, Eur. Pharmacol. 471: 223-228).
• the interleukin-4 mice have been genetically manipulated to express high level of this Th2-bias cytokines in the skin. These mice develop skin lesions and pruritus with clinical features resembling those of human atopic dermatitis (Chan, L.S.
• mice expressing high level of the Th2-bias cytokine IL-13 develop skin inflammation bearing the hallmarks of atopic dermatitis such as skin lesions, epidermal barrier breakdown and intense pruritus (Zheng, T. et al., J. Invest. Dermatol. 2008, Oct 16, online publication).
• MRL lpr mice have systemic autoimmunity resulting from a mutation in the lymphocyte death promoting Fas gene. Spontaneous scratching and skin lesions develop in MRL/lpr mice at 18 weeks of age (Umeuchi, H et al 2005, Eur J Pharm.
• mice that have already shown scratching behavior can be video recorded for one hour (or other length of time) to establish an itch intensity baseline.
• the test compound can be administered orally or by other routes. Mice can be video recorded to determine the frequency of scratching.
• the efficacy of a test compound can be compared to a sham treatment or a positive control.
• a model for canine atopic dermatitis has been utilized through an environmental house dust mite challenge of high-IgE-producing beagles, mite hypersensitive dogs with atopic dermatitis and normal dogs (Vet Dermatol. 2006 Feb;17(l):24-35).
• This model utilized high-IgE beagles epicutaneously sensitized to house dust mite (HDM) as a model for cAD.
• HDM house dust mite
• Six high-IgE beagles were environmentally challenged with HDM using various doses and protocols. Similar challenge protocols were used in positive and negative control dogs: three dogs with naturally occurring cAD and positive intradermal skin test (IDT) to HDM and three normal dogs without history of skin disease and negative IDT to HDM.
• IDT intradermal skin test
• a guinea pig pruritus model (Exp Dermatol. 2002 Aug;l 1(4):285-91) has been described using iontophoresis of histamine and capsaicin. It was also shown that contact sensitization with 2-4 dinitrochlorobenzene (DNCB) can be used as a simple assay for chronic itch allowing study of scratching over at least a 15-h period.
• DNCB dinitrochlorobenzene
• a primate pruritus model (J Neurosci. 2008 Jul 23;28(30):7659-69) has been described.
• the existence of two peripheral pathways for itch has been suggested in some species: one pathway that is responsive to histamine and a second pathway that can be activated by nonhistaminergic pruritogens (e.g., cowhage spicules).
• nonhistaminergic pruritogens e.g., cowhage spicules.
• behavioral responses and neuronal activity in unmyelinated afferent fibers can be assessed in monkey after topical application of cowhage spicules or intradermal injection of histamine and capsaicin.
• histamine induced itch can be analyzed in humans by injecting a pruritogen into the skin by iontophoresis.
• a fresh solution of histamine in saline can be prepared shortly before the experiment.
• a cotton disk can be soaked with this solution and mounted into the application chamber of an iontophoresis applicator.
• An anodal current and a charge of 20 mC (1 mA, 20 s) can be used to deliver histamine iontophoretically into the skin.
• the intensity of itch can be graded by subjects on a 100mm visual analogue scale (VAS) at 20s intervals for 5min after the injection of histamine.
• VAS 100mm visual analogue scale
• Compounds may be further selected based on their peripheral restriction (or lack thereof).
• One aspect of the present invention relates to H3R antagonists that are peripherally restricted.
• One aspect of the present invention relates to anti-pruritic agents that are peripherally restricted.
• the Caco-2 cell line was purchased from the American Type Culture Collection (ATCC) (Manassas, VA) and grown with Minimum Essential Medium supplemented with 10% fetal bovine serum, 100 units/mL penicillin, 100 ⁇ g/mL streptomycin, and 1% nonessential amino acids at 37°C in 5% C0 2 .
• Multi-well insert systems with an array of 12 or 24 individual inserts were used in the permeability assay. Each multi-well insert unit has two compartments: the top compartment and the bottom compartment. The top compartment is the insert and is commonly referred to as the apical compartment or apical side (A).
• the bottom compartment is the well of a multi-well plate where the insert is placed and is referred to as the basal compartment or basal side (B).
• Basal compartment or basal side (B).
• Caco-2 cells were seeded on a porous filter membrane. Cell culture medium was changed every two days. Cells were used for the transport experiments between 24 to 26 days after seeding.
• AQ/At is the appearance rate (DPM/sec of radiolabeled test compound; umol/sec of non-labeled test compound) on the receiver side during the permeation process
• A is the surface area of the cell monolayers
• Co is the initial concentration (DPM/mL of radiolabeled test compound; umol/mL of non-labeled test compound) on the donor side.
• Efflux ratio was defined as the ratio of Papp in the basolateral to apical direction over the Papp in the apical to basolateral direction.
• Brain vs Plasma concentrations (or ratio) for administered compounds. For example this may be calculated based on standard curve concentration. After detection by LC/MS/MS, the final brain concentrations can be calculated. Plasma sample concentrations are calculated based on standard curve concentration. Brain/Plasma ratio can be calculated with the final brain and plasma data of each animal.
• compounds are further selected based on desirable pharmacokinetic properties.
• the compound would exhibit a Ki ⁇ 10 nM across species.
• the compound exhibits selectivity across a broad receptor panel. In another embodiment, the compound does not exhibit CYP or hERG liability.
• the compound is efficacious in a histamine-mediated itch model. In another embodiment, the compound is efficacious in a dermatitis-driven itch model. In another embodiment, the compound has at least 10X potency separation between wake promotion and itch models.
• the compound or agent is restricted in some manner from crossing the blood brain barrier. In another embodiment, the compound or agent may be less than 100% restricted to the periphery. In one embodiment, the compound or agent has a braimplasma ratio (B/P) which will be less than 1 in at least one species.
• B/P braimplasma ratio
• the H3R antagonist or anti -pruritic agent has a brain to plasma ratio of about 0.5 or less. In another embodiment, the H3R antagonist or anti-pruritic agent has a brain to plasma ratio of about 0.4 or less. In another embodiment, the H3R antagonist or antipruritic agent has a brain to plasma ratio of about 0.3 or less. In another embodiment, the H3R antagonist or anti-pruritic agent has a brain to plasma ratio of about 0.2 or less. In another embodiment, the H3R antagonist or anti-pruritic agent has a brain to plasma ratio of about 0.15 or less. In another embodiment, the H3R antagonist or anti-pruritic agent has a brain to plasma ratio of about 0.1 or less. In another embodiment, the H3R antagonist or anti-pruritic agent has a brain to plasma ratio of about 0.01 or less.
• the H3R antagonist or anti-pruritic agent has a brain to plasma ratio of about 0.5 or less after administration in a pruritus model animal. In another embodiment, the H3R antagonist or anti-pruritic agent has a brain to plasma ratio of about 0.4 or less after administration in a pruritus model animal. In another embodiment, the H3R antagonist or anti- pruritic agent has a brain to plasma ratio of about 0.3 or less after administration in a pruritus model animal. In another embodiment, the H3R antagonist or anti-pruritic agent has a brain to plasma ratio of about 0.2 or less after administration in a pruritus model animal.
• the H3R antagonist or anti-pruritic agent has a brain to plasma ratio of about 0.15 or less after administration in a pruritus model animal. In another embodiment, the H3R antagonist or anti- pruritic agent has a brain to plasma ratio of about 0.1 or less after administration in a pruritus model animal. In another embodiment, the H3R antagonist or anti-pruritic agent has a brain to plasma ratio of about 0.01 or less after administration in a pruritus model animal.
• the compound has good oral bioavailability.
• the compound shows Tl/2>lh in microsomal stability across species.
• the compound shows in vivo Tl/2 in animal species consistent with a QD or BID regimen.
• the compound shows hERG > 10 uM.
• the compound is Ames negative.
• the compound has acceptable tolerability in rat repeat dosing.
• a H3R antagonist or anti-pruritic agent such as an antagonist or an inverse agonist, can be formulated into pharmaceutical compositions and medicaments for use in accordance with the present invention using techniques well known in the art.
• One aspect of the present invention relates to compositions comprising a H3R antagonist for use in treating pruritus/itch. Proper formulation is dependent on the route of administration chosen. In certain embodiments, the administration is to a non-human vertebrate or to a non-human mammal.
• the anti-pruritic agent/H3R antagonist is subsequently admixed with a pharmaceutical carrier.
• Pharmaceutical carriers are known in the art, such as pharmaceutical carriers described herein.
• the H3R antagonist is formulated as a composition.
• the H3R antagonist is a H3R inverse agonist.
• the anti-pruritic agent is subsequently admixed with a
• formulated compounds or agents can exhibit all the features previously described for compounds in relation to the screening assays or otherwise described in this application.
• compounds for formulation, method of treatment or any of the other embodiments described herein may, for example, demonstrate the selectivity, B/P ratios, or potency as previously or subsequently described.
• One aspect of the present invention relates to methods of preparing a pharmaceutical composition comprising a H3R antagonist for treating a condition characterized by itch, a method comprising:
• the H3R antagonist is an antagonist of the human H3R.
• the H3R antagonist is orally active.
• the H3R antagonist is a selective H3R antagonist.
• the H3R antagonist has a selectivity for H3R over H1R, H2R, or
• H4R of at least about 10 to fold.
• the H3R antagonist has an IC50 of less than about 1 ⁇ .
• the H3R antagonist has an IC50 of less than about 100 nM.
• the H3R antagonist has an IC50 of less than about 1 nM.
• the H3R antagonist is a small molecule.
• the mammal is a non-human mammal.
• the mammal is selected from the group consisting of a mouse, a rat, a dog and a non-human primate.
• the mammal is a human.
• the pharmaceutical composition is in a dosage form.
• the H3R antagonist has an IC50 of less than 1 ⁇ .
• the H3R antagonist has an IC50 of less than about 10 nM.
• the H3R antagonist has a selectivity for H3R over a H1R, H2R, or H4R of at least about 10 to fold.
• the dosage form is in combination with a H1R antagonist.
• the present invention provides a method of treating pruritus in a subject in need thereof by administering a therapeutically effective amount of a H3R antagonist.
• the H3R antagonist is in an amount sufficient to reduce itch in an individual.
• the H3R antagonist is selected from and the following compounds and pharmaceutically acceptable salts, solvates, and hydrates thereof:
• the invention comprises conjugates of antibodies, e.g. Fv, Fab, and F(ab)'2, bifunctional hybrid antibodies and single chain antibodies that are known to one skilled it the art.
• the method of treating pruritus in a subject in need thereof involves administering a therapeutically effective amount of a H3R antagonist in conjunction with a therapeutically effective amount of another compound known to facilitate the treatment of pruritus.
• the methods disclosed herein can be used in conjunction with other known anti-pruritus therapies such as menthol and phenol, calamine, topical
• antihistamines for uremia: dialysis, UVB phototherapy and for paraneoplasia: paroxetin.
• the methods disclosed herein can be used in conjunction with therapies for eczema, atopic eczematous dermatitis, seborrheic dermatitis, atopic dermatitis, contact dermatitis, irritant dermatitis, xerosis (dry skin), psoriasis, a fungal infection, athlete's foot, a yeast infection, diaper rash, vaginal itch, parasitic infections, parasitic infestations including scabies and lice, lichen planus, lichen simplex, lichen simplex chronicus, lichen sclerosis, itch secondary to medications, senile itch, uremia, idiopathic itch, itch associated with liver cirrhosis, itch associated with inflammation, itch associated with allergies, itch associated with cancer, itch associated with chemotherapy, itch associated with kidney disease, itch associated with haemodialysis, burns, scalds, sunburn, wound healing, itch associated with an insect bite, itch associated
• compositions include solutions, suspensions, lotions, gels, creams, ointments, emulsions, skin patches, etc. All of these dosage forms, along with methods for their preparation, are well known in the pharmaceutical and cosmetic art: Harry's Cosmeticology (Chemical Publishing, 7th ed. 1982); Remington's Pharmaceutical Sciences (Mack Publishing Co., 18th ed. 1990).
• topical formulations contain the active ingredient in a concentration range of 0.001 to 10 mg/mL, in admixture with suitable vehicles.
• Other desirable ingredients for use in such anti- pruritic preparations include preservatives, co-solvents, viscosity building agents, carriers, etc.
• the carrier itself or a component dissolved in the carrier may have palliative or therapeutic properties of its own, including moisturizing, cleansing, or anti-inflammatory/anti-itching properties.
• the H3R antagonists can be combined with a therapeutically effective amounts of anti-inflammation agents such as corticosteroids, fungicides, antibiotics, moisturizers or anti- itching compounds.
• Penetration enhancers may, for example, be surface active agents; certain organic solvents, such as dimethyl sulfoxide and other sulfoxides, dimethyl-acetamide and pyrrolidone; certain amides of heterocyclic amines, glycols (e.g. propylene glycol); propylene carbonate; oleic acid; alkyl amines and derivatives; various cationic, anionic, nonionic, and amphoteric surface active agents; and the like.
• organic solvents such as dimethyl sulfoxide and other sulfoxides, dimethyl-acetamide and pyrrolidone
• certain amides of heterocyclic amines such as glycols (e.g. propylene glycol); propylene carbonate; oleic acid; alkyl amines and derivatives; various cationic, anionic, nonionic, and amphoteric surface active agents; and the like.
• Topical administration of a pharmacologically effective amount may utilize transdermal delivery systems well known in the art.
• the H3R antagonists may also be administered systemically, such as oral, parenteral, nasal inhalation, and intrarectal are also contemplated.
• additional conventional pharmaceutical preparations such as tablets, granules, powders, capsules, and sprays may be preferentially required.
• further conventional additives such as binding-agents, wetting agents, propellants, lubricants, and stabilizers may also be required.
• Systemic administration preferably comprises ingestion of any solid or solution carriers containing a pharmacologically effective amount of one or more of the H3R antagonists.
• Such solid or solution carriers may comprise pills, hard tablets, soft tablets, gums or ordinary liquids.
• systemic administration of a pharmacologically effective amount may comprise invasive methodologies including intravenous, subcutaneous, intramuscular or intralesional injection of a suitable carrier, such as saline, containing a pharmacologically effective amount of one or more of the H3R antagonists.
• the route of administration, dosage form, and the effective amount vary according to the potency of the selected H3R antagonist, its physicochemical characteristics, and according to the location of itch sensations.
• the selection of proper dosage is well within the skill of an ordinary skilled physician.
• Topical formulations are usually administered up to four-times a day.
• H3R antagonists as anti -pruritics may be combined with, for example, Hl- antihistamines to provide superior therapy via additive or synergistic interaction.
• the itch may be associated with a disease or disorder related from the group consisting of eczema, atopic eczematous dermatitis, seborrheic dermatitis, atopic dermatitis, lichen planus, senile itch, uremia, idiopathic itch, itch associated with liver cirrhosis, itch associated with inflammation, itch associated with allergies, itch associated with cancer, itch associated with haemodialysis, burns, scalds, sunburn, insect bites, urticaria, sweat gland abnormalities, bullous pemphigoid, photodermatoses, skin blisters, adult acne, chicken pox, and dermatitis herpetiformis.
• H3R antagonists of the present invention can be administered in any suitable way.
• Suitable routes of administration include oral, nasal, rectal, transmucosal, transdermal, or intestinal administration, parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intrapulmonary (inhaled) or intraocular injections using methods known in the art.
• Other suitable routes of administration are aerosol and depot formulation. Sustained release formulations, particularly depot, of the invented medicaments are expressly contemplated.
• the compounds according to the present invention are administered orally.
• the compounds according to the present invention can be made up in solid or liquid form, such as tablets, capsules, powders, syrups, elixirs and the like, aerosols, sterile solutions, suspensions or emulsions, and the like.
• the H3R antagonist is administered orally.
• Formulations for oral administration may be in the form of aqueous solutions and suspensions, in addition to solid tablet and capsule formulations.
• the aqueous solutions and suspensions may be prepared from sterile powders or granules.
• the compounds may be dissolved in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, and/or various buffers.
• Other adjuvants are well and widely known in the art.
• compositions of the H3R antagonist may be prepared by methods well known in the art, e.g., by means of conventional mixing, dissolving, granulation, dragee-making, levigating, emulsifying, encapsulating, entrapping, lyophilizing processes or spray drying.
• compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
• suitable pharmaceutically acceptable carriers are available to those in the art (see, e.g., Remington: The Science and Practice of Pharmacy, (Gennaro et al., eds.), 20 th Edition, 2000, Lippincott Williams & Wilkins; and Handbook of Pharmaceutical Excipients (Rowe et al., eds), 4 th Edition, 2003, Pharmaceutical Press). Proper formulation is dependent upon the route of administration chosen.
• carrier material or “excipient” material herein means any substance, not itself a therapeutic agent, used as a carrier and/or dilutent and/or adjuvant, or vehicle for delivery of a therapeutic agent to a subject or added to a pharmaceutical composition to improve its handling or storage properties or to permit or facilitate formation of a dose unit of the composition into a discrete article such as a capsule or tablet suitable for oral administration.
• Excipients can include, by way of illustration and not limitation, diluents, disintegrants, binding agents, adhesives, wetting agents, polymers, lubricants, glidants, substances added to mask or counteract a disagreeable taste or odor, flavors, dyes, fragrances, and substances added to improved appearance of the composition.
• Acceptable excipients include stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, magnesium carbonate, talc, gelatin, acacia gum, sodium alginate, pectin, dextrin, mannitol, sorbitol, lactose, sucrose, starches, gelatin, cellulosic materials, such as cellulose esters of alkanoic acids and cellulose alkyl esters, low melting wax cocoa butter or powder, polymers, such as polyvinylpyrrolidone, polyvinyl alcohol, and polytheylene glycols, and other pharmaceutically acceptable materials.
• the components of the pharmaceutical composition can be encapsulated or tableted for convenient administration.
• One aspect of the present invention relates to methods for treating pruritus in an individual comprising administering to the individual in need thereof a therapeutically effective amount of a H3R antagonist.
• H3R antagonist or a pharmaceutical composition as described herein.
• One aspect of the present invention relates to methods for treating pruritus in an individual comprising administering to the individual in need thereof a therapeutically effective amount of a H3R antagonist, wherein the H3R antagonist is identified by using any of the methods described herein.
• the H3R antagonist is administered in the form of a pharmaceutical composition comprising a therapeutically effective amount of said H3R antagonist as active ingredient, in admixture with a pharmaceutical carrier.
• One aspect of the present invention relates to uses of a H3R antagonist in the manufacture of a medicament for treating pruritus.
• One aspect of the present invention relates to uses of a H3R antagonist in the manufacture of a medicament for treating pruritus, wherein the H3R antagonist is identified by using any of the methods described herein.
• One aspect of the present invention relates to H3R antagonists for use in a method for the treatment of pruritus.
• One aspect of the present invention relates to H3R antagonists for use in a method for the treatment of pruritus, wherein the H3R antagonist is identified by using any of the methods described herein.
• the H3R antagonist is administered in the form of a pharmaceutical composition comprising a therapeutically effective amount of said H3R antagonist as active ingredient, in admixture with a pharmaceutical carrier.
• One aspect of the present invention relates to H3R antagonists for use in a method for treating pruritus.
• the H3R antagonist is administered in the form of a pharmaceutical composition comprising a therapeutically effective amount of said H3R antagonist as active ingredient, in admixture with a pharmaceutical carrier.
• the H3R antagonist is applied topically.
• the H3R antagonist is applied topically to the site afflicted with itch.
• the H3R antagonist is applied topically and has an IC50 (H3R) of about 100 nM or less.
• the pharmaceutical composition is formulated in a form suitable for topical application.
• the H3R antagonist is administered systemically.
• the method further comprises administering a therapeutic amount of a H1R antagonist.
• the antagonist is administered orally.
• the H3R antagonist is peripherally restricted.
• Pharmaceutically acceptable refers to those properties and/or substances which are acceptable to the patient from a pharmacological/toxicological point of view and to the manufacturing pharmaceutical chemist from a physical/chemical point of view regarding composition, formulation, stability, patient acceptance and bioavailability.
• Dragee cores are provided with suitable coatings.
• suitable coatings may be used which may optionally contain gum Arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
• Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
• compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
• the push-fit capsules can contain the active ingredients in admixture with a filler such as lactose, a binder such as starch, and/or a lubricant such as talc or magnesium stearate and, optionally, stabilizers.
• the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, liquid polyethylene glycols, cremophor, capmul, medium or long chain mono-, di- or triglycerides.
• Stabilizers may be added in these formulations, also.
• a H3R antagonist may be delivered using a sustained-release system.
• sustained-release materials have been established and are well known to those skilled in the art.
• Sustained-release tablets or capsules are particularly preferred.
• a time delay material such as glyceryl monostearate or glyceryl distearate may be employed.
• the dosage form may also be coated by the techniques described in the U.S. Pat. Nos. 4,256,108, 4, 166,452, and 4,265,874 to form osmotic therapeutic tablets for controlled release.
• therapies of the present invention may be administered or provided alone or in combination with one or more other pharmaceutically or physiologically acceptable compound.
• the other pharmaceutically or physiologically acceptable compound i.e., second pharmaceutical agent
• the present invention features a composition comprising or consisting essentially of an amount of a H3R antagonist according to the present invention. In one aspect, the present invention features a pharmaceutical composition comprising or consisting essentially of an amount of a H3R antagonist according to the present invention and at least one pharmaceutically acceptable carrier.
• the present invention relates to a composition
• a composition comprising a compound identified according to one or more of the screening methods described herein.
• the present invention features a composition comprising or consisting essentially of an amount of a H3R antagonist according to the present invention.
• the present invention features a pharmaceutical composition comprising or consisting essentially of an amount of a H3R antagonist according to the present invention and at least one pharmaceutically acceptable carrier.
• the present invention also relates to a dosage form of the composition or of the pharmaceutical composition wherein the H3R antagonist is in an amount sufficient to give an effect in treating a condition characterized by itch.
• the H3R antagonist is in an amount sufficient to reduce itch in an individual.
• compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an amount to achieve their intended purpose.
• a pharmaceutical composition of the present invention is understood to be useful for treating a condition characterized by itch, such as pruritus.
• determination of the amount of a H3R antagonist sufficient to achieve an intended purpose according to the invention is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
• the data obtained from animal studies including but not limited to studies using mice, rats, rabbits, pigs, and non-human primates, can be used in formulating a range of dosage for use in humans.
• one skilled in the art understands how to extrapolate in vivo data obtained in an animal model system to another, such as a human. In some circumstances, these extrapolations may merely be based on the weight of the animal model in comparison to another, such as a human; in other circumstances, these extrapolations are not simply based on weights but rather incorporate a variety of factors.
• compositions of this invention are selected in accordance with a variety factors as cited above.
• the actual dosage regimen employed may vary widely and therefore may deviate from a preferred dosage regimen and one skilled in the art will recognize that dosage and dosage regimen outside these typical ranges can be tested and, where appropriate, may be used in the methods of this invention.
• An exemplary animal model system is mouse.
• Dosage amount and interval may be adjusted in order to provide an intended therapeutic effect. It will be appreciated that the exact dosage of a H3R antagonist in accordance with the present invention will vary depending on the H3R antagonist, its potency, the mode of administration, the age and weight of the patient and the severity of the condition to be treated. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.
• an amount of a H3R antagonist in accordance with the present invention is less than about 0.001 mg/kg body weight, less than about 0.005 mg kg body weight, less than about 0.01 mg/kg body weight, less than about 0.05 mg/kg body weight, less than about 0.1 mg/kg body weight, less than about 0.5 mg/kg body weight, less than about 1 mg/kg body weight, less than about 5 mg/kg body weight, less than about 10 mg/kg body weight, less than about 50 mg/kg body weight, or less than about 100 mg/kg body weight.
• an amount of a H3R antagonist in accordance with the present invention is less than about 0.001-100 mg/kg body weight, less than about 0.001-50 mg/kg body weight, less than about 0.001-10 mg/kg body weight, less than about 0.001-5 mg/kg body weight, less than about 0.001-1 mg/kg body weight, less than about 0.001 to 0.5 mg/kg body weight, less than about 0.001-0.1 mg/kg body weight, less than about 0.001-0.05 mg/kg body weight, less than about 0.001-0.01 mg/kg body weight, or less than about 0.001-0.005 mg/kg body weight.
• Dosage amount and interval may be adjusted individually to provide plasma levels of a H3R antagonist according to the present invention which achieve an intended therapeutic effect. Dosage intervals can also be determined using the value for a selected range of H3R antagonist concentration so as to achieve the intended therapeutic effect.
• a H3R antagonist should be administered using a regimen that maintains plasma levels within the selected range of a H3R antagonist concentration for 10-90% of the time, preferably between 30-99% of the time, and most preferably between 50-90% of the time. In cases of local administration or selective uptake, the range of H3R antagonist concentration providing the intended therapeutic effect may not be related to plasma concentration.
• composition administered will, of course, be dependent on the individual being treated, on the individual's weight, the severity of the affliction, the manner of administration, and the judgment of the prescribing physician.
• the present invention accordingly features a method of treating a condition characterized by itch, such as pruritus, comprising administering to an individual in need thereof a therapeutically effective amount of a composition comprising or consisting essentially of an amount of a H3R antagonist according to the present invention.
• the composition is a pharmaceutical composition.
• active ingredient is defined in the context of a
• pharmaceutical composition and is intended to mean a component of a pharmaceutical composition that provides the primary pharmacological effect, as opposed to an "inactive ingredient” which would generally be recognized as providing no pharmaceutical benefit.
• the amount of active ingredient, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of
• compositions of this invention are selected in accordance with a variety factors as cited above.
• the actual dosage regimen employed may vary widely and therefore may deviate from a preferred dosage regimen and one skilled in the art will recognize that dosage and dosage regimen outside these typical ranges can be tested and, where appropriate, may be used in the methods of this invention.
• the desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day.
• the sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations.
• the daily dose can be divided, especially when relatively large amounts are administered as deemed appropriate, into several, for example 2, 3 or 4 part administrations. If appropriate, depending on individual behavior, it may be necessary to deviate upward or downward from the daily dose indicated.
• the compounds of the present invention can be administrated in a wide variety of oral and parenteral dosage forms. It will be obvious to those skilled in the art that the following dosage forms may comprise, as the active component, either a compound of the invention or a pharmaceutically acceptable salt, solvate or hydrate of a compound of the invention.
• a suitable pharmaceutically acceptable carrier can be either solid, liquid or a mixture of both.
• Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories and dispersible granules.
• a solid carrier can be one or more substances which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.
• the carrier is a finely divided solid which is in a mixture with the finely divided active component.
• the active component is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted to the desire shape and size.
• the powders and tablets may contain varying percentage amounts of the active compound.
• a representative amount in a powder or tablet may contain from 0.5 to about 90 percent of the active compound; however, an artisan would know when amounts outside of this range are necessary.
• Suitable carriers for powders and tablets are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium
• preparation is intended to include the formulation of the active compound with encapsulating material as carrier providing a capsule in which the active component, with or without carriers, is surrounded by a carrier, which is thus in association with it.
• carrier which is thus in association with it.
• cachets and lozenges are included. Tablets, powders, capsules, pills, cachets and lozenges can be used as solid forms suitable for oral administration.
• a low melting wax such as an admixture of fatty acid glycerides or cocoa butter
• the active component is dispersed homogeneously therein, as by stirring.
• the molten homogenous mixture is then poured into convenient sized molds, allowed to cool and thereby to solidify.
• Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or sprays containing in addition to the active ingredient such carriers as are known in the art to be appropriate.
• Liquid form preparations include solutions, suspensions and emulsions, for example, water or water-propylene glycol solutions.
• parenteral injection liquid preparations can be formulated as solutions in aqueous polyethylene glycol solution.
• injectable preparations for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
• the sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.
• Suitable 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 find use in the preparation of injec tables.
• the compounds according to the present invention may thus be formulated for parenteral administration (e.g. by injection, for example bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative.
• the pharmaceutical compositions 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.
• the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g. sterile, pyrogen-free water, before use.
• Aqueous formulations suitable for oral use can be prepared by dissolving or suspending the active component in water and adding suitable colorants, flavors, stabilizing and thickening agents, as desired.
• Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, or other well-known suspending agents.
• viscous material such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, or other well-known suspending agents.
• solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for oral administration.
• liquid forms include solutions, suspensions and emulsions.
• These preparations may contain, in addition to the active component, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents and the like.
• the compounds according to the invention may be formulated as ointments, creams or lotions, or as a transdermal patch.
• Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents.
• Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents.
• Formulations suitable for topical administration in the mouth include lozenges comprising active agent in a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
• Solutions or suspensions are applied directly to the nasal cavity by conventional means, for example with a dropper, pipette or spray.
• the formulations may be provided in single or multi-dose form. In the latter case of a dropper or pipette, this may be achieved by the patient administering an appropriate, predetermined volume of the solution or suspension. In the case of a spray, this may be achieved for example by means of a metering atomizing spray pump.
• Administration to the respiratory tract may also be achieved by means of an aerosol formulation in which the active ingredient is provided in a pressurized pack with a suitable propellant.
• aerosol formulation in which the active ingredient is provided in a pressurized pack with a suitable propellant.
• the compounds of the present invention or pharmaceutical compositions comprising them are administered as aerosols, for example as nasal aerosols or by inhalation, this can be carried out, for example, using a spray, a nebulizer, a pump nebulizer, an inhalation apparatus, a metered inhaler or a dry powder inhaler.
• Pharmaceutical forms for administration of the compounds of the present invention as an aerosol can be prepared by processes well known to the person skilled in the art.
• solutions or dispersions of the compounds of the present invention in water, water/alcohol mixtures or suitable saline solutions can be employed using customary additives, for example benzyl alcohol or other suitable preservatives, absorption enhancers for increasing the bioavailability, solubilizers, dispersants and others and, if appropriate, customary propellants, for example include carbon dioxide, CFCs, such as,
• the aerosol may conveniently also contain a surfactant such as lecithin.
• the dose of drug may be controlled by provision of a metered valve.
• the compound In formulations intended for administration to the respiratory tract, including intranasal formulations, the compound will generally have a small particle size for example of the order of 10 microns or less. Such a particle size may be obtained by means known in the art, for example by micronization. When desired, formulations adapted to give sustained release of the active ingredient may be employed.
• the active ingredients may be provided in the form of a dry powder, for example, a powder mix of the compound in a suitable powder base such as lactose, starch, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidone (PVP).
• a powder mix of the compound in a suitable powder base such as lactose, starch, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidone (PVP).
• PVP polyvinylpyrrolidone
• the powder carrier will form a gel in the nasal cavity.
• the powder composition may be presented in unit dose form for example in capsules or cartridges of, e.g., gelatin, or blister packs from which the powder may be administered by means of an inhaler.
• the pharmaceutical preparations are preferably in unit dosage forms.
• the preparation is subdivided into unit doses containing appropriate quantities of the active component.
• the unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules and powders in vials or ampoules.
• the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.
• Tablets or capsules for oral administration and liquids for intravenous administration are preferred compositions.
• the compounds according to the invention may optionally exist as pharmaceutically acceptable salts including pharmaceutically acceptable acid addition salts prepared from pharmaceutically acceptable non-toxic acids including inorganic and organic acids.
• Representative acids include, but are not limited to, acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethenesulfonic, dichloroacetic, formic, fumaric, gluconic, glutamic, hippuric, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, oxalic, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, oxalic, p-toluenesulfonic and the like.
• Certain compounds of the present invention which contain a carboxylic acid functional group may optionally exist as pharmaceutically acceptable salts containing non-toxic, pharmaceutically acceptable metal cations and cations derived from organic bases.
• Representative metals include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium, zinc and the like. In some embodiments the pharmaceutically acceptable metal is sodium.
• Organic bases include, but are not limited to, benzathine ( N2-dibenzylemane-l,2-diamine), chloroprocaine (2- (diethylamino)ethyl 4-(chloroamino)benzoate), choline, diethanolamine, ethylenediamine, meglumine ((2R,3R,4R,55)-6-(methylamino)hexane-l,2,3,4,5-pentaol), procaine (2- (diethylamino)ethyl 4-aminobenzoate), and the like.
• Certain pharmaceutically acceptable salts are listed in Berge, et al., Journal of Pharmaceutical Sciences, 66: 1-19 (1977).
• the acid addition salts may be obtained as the direct products of compound synthesis.
• the free base may be dissolved in a suitable solvent containing the appropriate acid and the salt isolated by evaporating the solvent or otherwise separating the salt and solvent.
• the compounds of this invention may form solvates with standard low molecular weight solvents using methods known to the skilled artisan.
• Pro-drugs refers to compounds that have been modified with specific chemical groups known in the art and when administered into an individual these groups undergo biotransformation to give the parent compound. Pro-drugs can thus be viewed as compounds of the invention containing one or more specialized non-toxic protective groups used in a transient manner to alter or to eliminate a property of the compound. In one general aspect, the "pro-drug” approach is utilized to facilitate oral absorption.
• T. Higuchi and V. Stella Pro-drugs as Novel Delivery Systems Vol. 14 of the A.C.S. Symposium Series; and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.
• composition for "combination-therapy” comprising admixing at least one compound according to any of the compound embodiments disclosed herein, together with at least one known pharmaceutical agent as described herein and a pharmaceutically acceptable carrier.
• H3R antagonists can be co-administered with other therapeutic agents to meet the treatment objectives.
• a H3R antagonist may be combined with antagonists of other histamine receptor subtypes, particularly H1R and H4R antagonists.
• H1R antagonists may include both sedation and non-sedation antihistamines such as diphenhydramine, cetirizine, hydroxyzine, chlorpheniramine, promethazine, fexofenadine, loratadine, and desloratadine.
• the composition further comprises an H1R antagonist in an amount sufficient to reduce itch in an individual.
• H4R antagonists to be combined may include compounds reported in the literature.
• a H3R antagonist with therapeutic agents currently used to treat itch in patients with chronic metabolic diseases.
• These drugs may include the bile resin cholestyramine and the antibiotic rifampicin, or opiate receptor antagonists naloxone, nalmefene and naltrexone.
• opiate receptor antagonists naloxone, nalmefene and naltrexone.
• H3R antagonists to be combined may include compounds reported in the literature.
• H3R receptor modulators are utilized as active ingredients in a pharmaceutical composition, these are not intended for use only in humans, but in other non-human mammals as well. Indeed, recent advances in the area of animal health-care mandate that consideration be given for the use of active agents, such as H3R receptor modulators, for the treatment of a H3R-associated disease or disorder in companionship animals (e.g., cats, dogs, etc.) and in livestock animals (e.g., cows, chickens, fish, etc.) Those of ordinary skill in the art are readily credited with understanding the utility of such compounds in such settings.
• companionship animals e.g., cats, dogs, etc.
• livestock animals e.g., cows, chickens, fish, etc.
• the dosage forms described herein may comprise, as the active component, either a compound described herein or a pharmaceutically acceptable salt or as a pharmaceutically acceptable solvate or hydrate thereof.
• various hydrates and solvates of the compounds described herein and their salts will find use as intermediates in the manufacture of pharmaceutical compositions. Typical procedures for making and identifying suitable hydrates and solvates, outside those mentioned herein, are well known to those in the art; see for example, pages 202-209 of K.J. Guillory, "Generation of Polymorphs, Hydrates, Solvates, and Amorphous Solids," in: Polymorphism in Pharmaceutical Solids, ed. Harry G. England, Vol.
• one aspect of the present invention pertains to methods of administering hydrates and solvates of compounds described herein and/or their pharmaceutical acceptable salts, that can be isolated and characterized by methods known in the art, such as, thermogravimetric analysis (TGA), TGA-mass spectroscopy, TGA-Infrared spectroscopy, powder X-ray diffraction (XRPD), Karl Fisher titration, high resolution X-ray diffraction, and the like.
• TGA thermogravimetric analysis
• TGA-mass spectroscopy TGA-Infrared spectroscopy
• powder X-ray diffraction (XRPD) powder X-ray diffraction
• Karl Fisher titration high resolution X-ray diffraction
• the present disclosure includes all isotopes of atoms occurring in the present compounds, intermediates, salts and crystalline forms thereof.
• Isotopes include those atoms having the same atomic number but different mass numbers.
• One aspect of the present invention includes every combination of one or more atoms in the present compounds, intermediates, salts, and crystalline forms thereof that is replaced with an atom having the same atomic number but a different mass number.
• One such example is the replacement of an atom that is the most naturally abundant isotope, such as H or 12 C, found in one the present compounds, intermediates, salts, and crystalline forms thereof, with a different atom that is not the most naturally abundant isotope, such as 2 H or 3 H (replacing 3 ⁇ 4), or n C, 13 C, or 14 C (replacing 12 C).
• a compound wherein such a replacement has taken place is commonly referred to as being an isotopically-labeled compound.
• Isotopic-labeling of the present compounds, intermediates, salts, and crystalline forms thereof can be accomplished using any one of a variety of different synthetic methods know to those of ordinary skill in the art and they are readily credited with understanding the synthetic methods and available reagents needed to conduct such isotopic-labeling.
• isotopes of hydrogen include 2 H (deuterium) and 3 H (tritium).
• Isotopes of carbon include n C, 13 C, and 14 C.
• Isotopes of nitrogen include 13 N and 15 N.
• Isotopes of oxygen include 15 0, 17 0, and 18 C.
• An isotope of fluorine includes 18 F.
• An isotope of sulfur includes 35 S.
• An isotope of chlorine includes 36 C1.
• Isotopes of bromine include 75 Br, 76 Br, 77 Br, and 82 Br.
• Isotopes of iodine include 123 1, 124 1, 125 I, and 131 I.
• Another aspect of the present invention includes compositions, such as, those prepared during synthesis, preformulation, and the like, and pharmaceutical compositions, such as, those prepared with the intent of using in a mammal for the treatment of one or more of the disorders described herein, comprising one or more of the present compounds, intermediates, salts, and crystalline forms thereof, wherein the naturally occurring distribution of the isotopes in the composition is perturbed.
• compositions and pharmaceutical compositions comprising compounds as described herein wherein the compound is enriched at one or more positions with an isotope other than the most naturally abundant isotope.
• Methods are readily available to measure such isotope perturbations or enrichments, such as, mass spectrometry, and for isotopes that are radio-isotopes additional methods are available, such as, radio-detectors used in connection with HPLC or GC.
• Example 1 Syntheses of Compounds of the Present Invention.
• TLC Thin-layer chromatography
• PK6F silica gel 60 A 1 mm plates (Whatman) and column chromatography was carried out on a silica gel column using Kieselgel 60, 0.063-0.200 mm (Merck). Evaporation was done under reduced pressure on a Biichi rotary evaporator.
• LCMS spec HPLC-pumps: LC-10AD VP, Shimadzu Inc.; HPLC system controller: SCL- 10A VP, Shimadzu Inc; UV-Detector: SPD-IOA VP, Shimadzu Inc; Autosampler: CTC HTS, PAL, Leap Scientific; Mass spectrometer: API 150EX with Turbo Ion Spray source, AB MDS Sciex; Software: Analyst 1.2.
• H3R antagonist of Formula (la) can be prepared according to procedures described in WO2008/005338, for example: 4'-[2-((R)-2-Methyl-pyrrolidin-l-yl)-ethyl]-biphenyl-4-sulfonic acid 4-chloro-benzylamide can be prepared according to Example 1.9 (Cmpd 24), WO2008/005338; 4'- [2-((R)-2-Methyl-pyrrolidin- 1 -yl)-ethyl] -biphenyl-4-sulfonic acid amide can be prepared according to Example 1.23 (Cmpd 35), WO2008/005338; 4'-[2-((R)-2-Methyl-pyrrolidin-l-yl)-ethyl]- biphenyl-4-sulfonic acid ethylamide can be prepared according to Example 1.21 (Cmpd 37), WO2008/005338; Propionic acid l-
• Propionic acid 2-(l- ⁇ 4'-[2-((R)-2-methyl-pyrrolidin-l-yl)-ethyl]-biphenyl-4- sulfonyl ⁇ -piperidin-4-yl)-ethyl ester can be prepared according to Example 2.24 (Cmpd 63), WO2008/005338; and Propionic acid l- ⁇ 4'-[2-((R)-2-methyl-pyrrolidin-l-yl)-ethyl]-biphenyl-4- sulfonyl ⁇ -piperidin-4-ylmethyl ester can be prepared according to Example 2.23 (Cmpd 68), WO2008/005338.
• H3R antagonist of Formula (Ila) can be prepared according to procedures described in WO2008/048609, for example: (R)-l-[2-(4'-Methanesulfonyl-biphenyl-4-yl)-ethyl]-2-methyl- pyrrolidine can be prepared according to Example 1.13 (Cmpd 1), WO2008/048609; (R)-l-[2-(4'- Ethanesulfonyl-biphenyl-4-yl)-ethyl]-2-methyl-pyrrolidine can be prepared according to Example 1.15 (Cmpd 2), WO2008/048609; (R)-l- ⁇ 2-[4'-(2-Methoxy-ethanesulfonyl)-biphenyl-4-yl]-ethyl ⁇ - 2-methyl-pyrrolidine can be prepared according to Example 1.5 (Cmpd 3), WO2008/048609; (R)- 2-Methyl-l- ⁇ 2-[4'-(propan
• H3R antagonist of Formula (Ilia) can be prepared according to procedures described in WO2009/058300, for example: (,S , )-4-((4'-(2-((R)-2-methylpyrrolidin-l-yl)ethyl)biphenyl-4- yl)methyl)oxazolidin-2-one can be prepared according to Example 1.26 (Cmpd 1),
• WO2009/058300; (,S , )-4-(4'-(2-((R)-2-methylpyrrolidin-l-yl)ethyl)biphenyl-4-yl)oxazolidin-2-one can be prepared according to Example 1.25 (Cmpd 2), WO2009/058300; (R)-4-((4'-(2-((R)-2- methylpyrrolidin-l-yl)ethyl)biphenyl-4-yl)methyl)oxazolidin-2-one can be prepared according to Example 1.4 (Cmpd 4), WO2009/058300; (R)-3-methyl-4-(4'-(2-((R)-2-methylpyrrolidin-l- yl)ethyl)biphenyl-4-yl)oxazolidin-2-one can be prepared according to Example 1.5 (Cmpd 5), WO2009/058300; (R)-3-isopropyl-4-(4'-
• H3R antagonist of Formula (IVa) can be prepared according to procedures described in WO2009/105206, for example: (R)-3-methoxy-l-(7-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)- 3,4-dihydroisoquinolin-2(l//)-yl)propan-l-one can be prepared according to Example 1.19 (Cmpd 1), WO2009/105206; (R)-cyclopropyl(6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)-3,4- dihydroisoquinolin-2(l//)-yl)mefhanone can be prepared according to Example 1.10 (Cmpd 3), WO2009/105206; (R)-cyclopropyl(5 4 2 2-methylpyrrolidin-l-yl)ethyl)phenyl)isoindolin-2- yl)methanone can
• Example 1.5-1 Preparation of Intermediate (/f)-6-(4-(2-(2-Methylpyrrolidin-l- yl)ethyl)phenyl)-l,2,3,4-tetrahydroisoquinoline.
• Step A Preparation of (/f)-l-(4-Bromophenethyl)-2-methylpyrrolidine.
• Step B Preparation of (/f)-4-(2-(2-Methylpyrrolidin-l-yl)ethyl)phenylboronic Acid.
• Step C Preparation of Intermediate (fl)-6-(4-(2-(2-Methylpyrrolidin-l- yl)ethyl)phenyl)-l,2,3,4-tetrahydroisoquinoline.
• 6-bromo-l,2,3,4-tetrahydroisoquinoline hydrochloride (2.00 g, 8.05 mmol)
• (R)-4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenylboronic acid 2.063 g, 8.85 mmol
• tetrakis(triphenylphosphine)palladium (0) 0.279 g, 0.241 mmol
• benzene (30.00 mL), ethanol (10.00 mL), and 2.0 M aqueous solution of sodium bicarbonate (8.05 mL, 16.09 mmol).
• the reaction mixture was refluxed for 6 h. Upon completion, water was added and the mixture was extracted with ethyl acetate. The organic layer was washed with brine, dried over Na 2 S0 4 , and concentrated. The residue was taken up in 1 M HC1 solution and washed with ethyl acetate. The aqueous layer was basified with 10% aqueous NaOH to pH ⁇ l 1, extracted with ethyl acetate, and concentrated. The residue was purified by silica gel column, eluting with 5-10% 2.0 M ammonia in methanol/DCM to give a yellow solid (1.20 g).
• Example 1.5-2 Preparation of (/f)-l-(6-(4-(2-(2-Methylpyrrolidin-l-yl)ethyl)phenyl)-3,4- dihydroisoquinolin-2(l/ )-yl)-2-(l/ -tetrazol-5-yl)ethanone (Compound 1).
• Example 1.5-3 Preparation of (/f)-4-(6-(4-(2-(2-Methylpyrrolidin-l-yl)ethyl)phenyl)-3,4- dihydroisoquinolin-2(l//)-yl)-4-oxobutanoic Acid (Compound 2).
• Example 1.5-4 Preparation of (/f)-2,2,3,3-tetrafluoro-4-(6-(4-(2-(2-methylpyrrolidin-l- yl)ethyl)phenyl)-3,4-dihydroisoquinolin-2(l/ )-yl)-4-oxobutanoic acid (Compound 8).
• Example 1.5-5 Preparation of (/f)-3,3-Dimethyl-5-(6-(4-(2-(2-methylpyrrolidin-l- yl)ethyl)phenyl)-3,4-dihydroisoquinolin-2(l/ )-yl)-5-oxopentanoic Acid (Compound 3).
• TFA salt of the title compound was prepared in a similar manner to the one described in Example 1.5-4 using (R)-6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)-l,2,3,4- tetrahydroisoquinoline dihydrochloride and 4,4-dimethyldihydro-2/i-pyran-2,6(3//)-dione.
• Example 1.5-6 Preparation of (/f)-2,2-Dimethyl-5-(6-(4-(2-(2-methylpyrrolidin-l- yl)ethyl)phenyl)-3,4-dihydroisoquinolin-2(l/ )-yl)-5-oxopentanoic Acid (Compound 4).
• TFA salt of the title compound was prepared in a similar manner to the one described in Example 1.5-4 using (R)-6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)-l,2,3,4- tetrahydroisoquinoline dihydrochloride and 3,3-dimethyldihydro-2/i-pyran-2,6(3H)-dione.
• LCMS m/z 463.5 [M+H] + .
• Example 1.5-7 Preparation of 3-Methyl-5-(6-(4-(2-((/f)-2-methylpyrrolidin-l- yl)ethyl)phenyl)-3,4-dihydroisoquinolin-2(l/ )-yl)-5-oxopentanoic Acid (Compound 5).
• TFA salt of the title compound was prepared in a similar manner to the one described in Example 1.5-4 using (R)-6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)-l,2,3,4- tetrahydroisoquinoline dihydrochloride and 4-methyldihydro-2/i-pyran-2,6(3//)-dione.
• LCMS m/z 449.3 [M+H] + .
• Example 1.5-8 Preparation of 3-hydroxy-5-(6-(4-(2-((/f)-2-methylpyrrolidin-l- yl)ethyl)phenyl)-3,4-dihydroisoquinolin-2(l/ )-yl)-5-oxopentanoic acid (Compound 6).
• TFA salt of the title compound was prepared in a similar manner to the one described in Example 1.5-4 using (R)-6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)-l,2,3,4- tetrahydroisoquinoline dihydrochloride and l,4-oxathiane-2,6-dione.
• LCMS m/z 453.5 [M+H] + .
• Example 1.5-10 Preparation of (/f)-2 ⁇ -Dimethyl-4-(6-(4-(2-(2-methylpyrrolidin-l- yl)ethyl)phenyl)-3,4-dihydroisoquinolin-2(l/ )-yl)-4-oxobutanoic Acid (Compound 9).
• TFA salt of the title compound was prepared in a similar manner to the one described in Example 1.5-4 using (R)-6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)-l,2,3,4- tetrahydroisoquinoline dihydrochloride and 3,3-dimethyldihydrofuran-2,5-dione.
• LCMS mJz 449.6 [M+H] + .
• Example 1.5-12 Preparation of (/f)-5-(6-(4-(2-(2-Methylpyrrolidin-l-yl)ethyl)phenyl)-3,4- dihydroisoquinolin-2(l/ )-yl)-5-oxopentanoic Acid (Compound 11).
• Example 1.5-13 Preparation of (fl)-Methyl 4-(6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)- 3,4-dihydroisoquinolin-2(l/ )-yl)-4-oxobutanoate (Compound 12).
• Example 1.5-15 Preparation of (/f)-(6-(4-(2-(2-Methylpyrrolidin-l-yl)ethyl)phenyl)-3,4- dihydroisoquinolin-2(l/ )-yl)(l/ -tetrazol-5-yl)methanone (Compound 14).

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