Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/06—Dipeptides
  • C07K5/06008—Dipeptides with the first amino acid being neutral
  • C07K5/06078—Dipeptides with the first amino acid being neutral and aromatic or cycloaliphatic
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/04—Centrally acting analgesics, e.g. opioids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• the invention described herein relates to peptide analogue compounds and the pharmaceutically acceptable salts of such compounds.
• the invention also relates to the processes for the preparation of the compounds, compositions containing the compounds, and the uses of such compounds and salts in treating diseases or conditions associated with opioid activity. More specifically the invention relates to the compounds and their salts useful as opioid agonists.
• the opioid receptor family consisting of ⁇ -, ⁇ - and ⁇ -receptors, is a member of the rhodopsin subfamily in the superfamily of G-protein coupled receptors (GPCRs). These receptors share extensive structural and sequence homology ( ⁇ 60% amino acid identity) but recognise structurally diverse ligands comprising exogenous opiates, endogenous peptides and synthetic peptidic and non-peptidic ligands (Waldhoer et al., Annu. Rev. Biochem (2004) 73: 953-990). Opiates e.g. morphine and synthetic opioids e.g.
• fentanyl acting through their activity at the opioid receptor are some of the most potent analgesic drugs for moderate to severe pain conditions.
• Opioid receptors are widely expressed in the central nervous system (CNS) and periphery of many species including man. Opioid receptors have been localised on peripheral processes of sensory neurones in animals and humans (Stein et al., 2003). Most opiates and opioids mediate their analgesic effects via peripheral, spinal and supraspinal receptors.
• Frakefamide a synthetic ⁇ -opioid receptor agonist demonstrated efficacy in a dental pain study (Becktor et al., abstract from 2002 World Congress on Pain) at doses that did not elicit respiratory depression ( ⁇ sterlund Modalen et al., abstract from 2002 World Congress on Pain; ⁇ sterlund Modalen et al., 2005; Anesth Analg, 100: 713-717).
• Synthetic compounds acting specifically through peripheral ⁇ -opioid receptors provide the potential to effectively manage pain without the centrally-mediated adverse effects of drugs like morphine, e.g. Current Pharmaceutical Design, 2004 (10), 743-757; Ther Clin Risk Manag. 2005 December; 1(4): 279-297.
• compounds should preferably bind potently to the ⁇ -opioid receptor whilst showing little affinity for other receptors and show functional activity as ⁇ -opioid receptor agonists. They should preferably be well absorbed from the gastrointestinal tract, and/or be injectable directly into the bloodstream, muscle, or subcutaneously, and/or be metabolically stable and possess favourable pharmacokinetic properties. When targeted against receptors in the central nervous system they should cross the blood brain barrier freely and when targeted selectively against receptors in the peripheral nervous system they should not cross the blood brain barrier. They should be non-toxic and demonstrate few side-effects. Furthermore, the ideal drug candidate will exist in a physical form that is stable, non-hygroscopic and easily formulated.
• the present invention relates to compounds of the Formula I:
• Ar 1 is phenyl optionally substituted by one or more substituents independently selected from OH, C 1-3 alkyl and halogen,
• Ar 1 is phenyl fused to a 5- or 6-membered heterocycle optionally substituted by one or more substituents independently selected from halogen, OH, and ⁇ O,
• Ar 2 is phenyl optionally substituted by one or more substituents independently selected from OH, C 1-3 alkyl and halogen,
• Ar 2 is phenyl fused to a 5- or 6-membered heterocycle optionally substituted by one or more substituents independently selected from halogen, OH, and ⁇ O,
• Gu is guanidinyl optionally substituted by up to 4 substituents independently selected from C 1-3 alkyl (or a tautomeric form thereof),
• R 1 is independently H or C 1-5 alkyl optionally substituted by Y,
• Y is halogen, S(O) m —(C 1-3 alkyl), CO 2 —R 5 or Gu,
• R 5 is H or C 1-6 alkyl
• R 2 is independently H or C 1-3 alkyl
• R 1 and R 2 together with the carbon atom to which they are attached can be a cyclopropyl group
• R 3 and R 4 is H and the other is Q where Q is H, NH 2 , NHC(O)(C 1-6 alkyl), Gu, NHC(O)(CH 2 ) n NH2, NHC(O)(CH 2 ) n Gu, or (CH 2 ) n Gu,
• n 1, 2, 3, 4 or 5
• the invention is also directed to pharmaceutical compositions comprising a therapeutically effective amount of a compound of formula I herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
• the invention is also directed to a method of treating a disease or condition indicated for treatment with an opioid receptor agonist, particularly a ⁇ -opioid receptor agonist, in a subject, by administering to a subject in need thereof a therapeutically effective amount of one or more of the compounds herein, or a pharmaceutically acceptable salt thereof.
• an opioid receptor agonist particularly a ⁇ -opioid receptor agonist
• the compounds of the present invention are potent agonists at the ⁇ -opioid receptor, and have a suitable PK profile to enable once daily dosing.
• the compounds of the present invention have physical properties that minimise penetration into the CNS.
• CNS penetration can be restricted through appropriate physiochemical properties such as lipophilicity, number of H-bond donors, polar surface area, number of charged centres (see e.g. K. M. M. Doan THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS 2002, Vol. 303 (3) 1029-1037 and L. Di, Expert Opinion on Drug Discovery June 2008, Vol. 3, No. 6: 677-687).
• treatment is potentially useful in the treatment of a range of disorders where an opioid agonist is indicated, particularly pain indications.
• treatment may include one or more of curative, palliative and prophylactic treatment.
• a compound of the present invention may be used to treat any physiological pain such as inflammatory pain, nociceptive pain, neuropathic pain, acute pain, chronic pain, musculo-skeletal pain, on-going pain, central pain, heart and vascular pain, head pain, orofacial pain.
• Other pain conditions which may be treated include intense acute pain and chronic pain conditions which may involve the same pain pathways driven by pathophysiological processes and as such cease to provide a protective mechanism and instead contribute to debilitating symptoms associated with a wide range of disease states.
• Pain There are a number of typical pain subtypes: 1) spontaneous pain which may be dull, burning, or stabbing; 2) pain responses to noxious stimuli are exaggerated (hyperalgesia); 3) pain is produced by normally innocuous stimuli (allodynia) (Meyer et al., 1994 Textbook of Pain 13-44). Pain can be divided into a number of different areas because of differing pathophysiology, these include nociceptive, inflammatory, neuropathic pain among others. It should be noted that some types of pain have multiple aetiologies and thus can be classified in more than one area, e.g. Back pain, Cancer pain have both nociceptive and neuropathic components.
• Nociceptive pain is induced by tissue injury or by intense stimuli with the potential to cause injury. Pain afferents are activated by transduction of stimuli by nociceptors at the site of injury and sensitise the spinal cord at the level of their termination. This is then relayed up the spinal tracts to the brain where pain is perceived (Meyer et al., 1994 Textbook of Pain 13-44).
• the activation of nociceptors activates two types of afferent nerve fibres. Myelinated A-delta fibres transmit rapidly and are responsible for the sharp and stabbing pain sensations, whilst unmyelinated C fibres transmit at a slower rate and convey the dull or aching pain.
• Moderate to severe acute nociceptive pain is a prominent feature of, but is not limited to pain from strains/sprains, post-operative pain (pain following any type of surgical procedure), posttraumatic pain, burns, myocardial infarction, acute pancreatitis, and renal colic. Also cancer related acute pain syndromes commonly due to therapeutic interactions such as chemotherapy toxicity, immunotherapy, hormonal therapy and radiotherapy.
• Moderate to severe acute nociceptive pain is a prominent feature of, but is not limited to, cancer pain which may be tumour related pain, (e.g. bone pain, headache and facial pain, viscera pain) or associated with cancer therapy (e.g.
• postchemotherapy syndromes chronic postsurgical pain syndromes, post radiation syndromes
• back pain which may be due to herniated or ruptured intervertabral discs or abnormalities of the lumbar facet joints, sacroiliac joints, paraspinal muscles or the posterior longitudinal ligament.
• a compound of the present invention can potentially be used to treat neuropathic pain and the symptoms of neuropathic pain including hyperalgesia, allodynia and ongoing pain.
• Neuropathic pain is defined as pain initiated or caused by a primary lesion or dysfunction in the nervous system (IASP definition). Nerve damage can be caused by trauma and disease and thus the term ‘neuropathic pain’ encompasses many disorders with diverse aetiologies. These include but are not limited to, Diabetic neuropathy, Post herpetic neuralgia, Back pain, Cancer neuropathy, HIV neuropathy, Phantom limb pain, Carpal Tunnel Syndrome, chronic alcoholism, hypothyroidism, trigeminal neuralgia, uremia, or vitamin deficiencies.
• Neuropathic pain is pathological as it has no protective role. It is often present well after the original cause has dissipated, commonly lasting for years, significantly decreasing a patients quality of life (Woolf and Mannion 1999 Lancet 353: 1959-1964).
• the symptoms of neuropathic pain are difficult to treat, as they are often heterogeneous even between patients with the same disease (Woolf & Decosterd 1999 Pain Supp. 6: S141-S147; Woolf and Mannion 1999 Lancet 353: 1959-1964). They include spontaneous pain, which can be continuous, or paroxysmal and abnormal evoked pain, such as hyperalgesia (increased sensitivity to a noxious stimulus) and allodynia (sensitivity to a normally innocuous stimulus).
• Intense acute pain and chronic pain may involve the same pathways driven by pathophysiological processes and as such cease to provide a protective mechanism and instead contribute to debilitating symptoms associated with a wide range of disease states. Pain is a feature of many trauma and disease states. When a substantial injury, via disease or trauma, to body tissue occurs the characteristics of nociceptor activation are altered. There is sensitisation in the periphery, locally around the injury and centrally where the nociceptors terminate. This leads to hypersensitivity at the site of damage and in nearby normal tissue. In acute pain these mechanisms can be useful and allow for the repair processes to take place and the hypersensitivity returns to normal once the injury has healed. However, in many chronic pain states, the hypersensitivity far outlasts the healing process and is normally due to nervous system injury.
• Chronic pain comprises one or more of, chronic nociceptive pain, chronic neuropathic pain, chronic inflammatory pain, breakthrough pain, persistent pain hyperalgesia, allodynia, central sensitisation, peripheral sensitisation, disinhibition and augmented facilitation.
• Chronic pain includes cancer pain, e.g. cancer pain arising from malignancy, adenocarcinoma in glandular tissue, blastoma in embryonic tissue of organs, carcinoma in epithelial tissue, leukemia in tissues that form blood cells, lymphoma in lymphatic tissue, myeloma in bone marrow, sarcoma in connective or supportive tissue, adrenal cancer, AIDS-related lymphoma, anemia, bladder cancer, bone cancer, brain cancer, breast cancer, carcinoid tumour s, cervical cancer, chemotherapy, colon cancer, cytopenia, endometrial cancer, esophageal cancer, gastric cancer, head cancer, neck cancer, hepatobiliary cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, Hodgkin's disease, lymphoma, non-Hodgkin's, nervous system tumours, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, thyroid
• Cancer pain also comprises visceral pain, e.g. visceral pain which arises from pancreatic cancer and/or metastases in the abdomen, somatic pain, e.g. somatic pain due to one or more of bone cancer, metastasis in the bone, postsurgical pain, sarcomas cancer of the connective tissue, cancer of bone tissue, cancer of blood-forming cells of the bone marrow, multiple myeloma, leukaemia, primary or secondary bone cancer.
• visceral pain e.g. visceral pain which arises from pancreatic cancer and/or metastases in the abdomen
• somatic pain e.g. somatic pain due to one or more of bone cancer, metastasis in the bone
• postsurgical pain sarcomas cancer of the connective tissue, cancer of bone tissue, cancer of blood-forming cells of the bone marrow, multiple myeloma, leukaemia, primary or secondary bone cancer.
• Inflammatory conditions include acute inflammation, persistent acute inflammation, chronic inflammation, and combined acute and chronic inflammation.
• Inflammatory pain includes acute inflammatory pain and/or chronic inflammatory pain wherein the chronic inflammatory pain can be pain involving both peripheral and central sensitisation and/or mixed etiology pain involving both inflammatory pain and neuropathic pain or nociceptive pain components.
• Inflammatory pain also comprises hyperalgesia, e.g. primary and/or secondary hyperalgesia. Additionally or alternatively the inflammatory pain can include allodynia.
• Inflammatory pain also comprises pain that persists beyond resolution of an underlying disorder or inflammatory condition or healing of an injury.
• Inflammatory pain is pain resulting an inflammatory condition. e.g. in response to acute tissue injury due to trauma, disease e.g. an inflammatory disease, immune reaction, the presence of foreign substances, chemicals or infective particles for example micro-organisms. Inflammatory conditions can be either acute or chronic inflammation or both.
• Inflammatory pain can result from an inflammatory condition due to an inflammatory disease such as inflammatory joint diseases, inflammatory connective tissue diseases, inflammatory autoimmune diseases, inflammatory myopathies, inflammatory digestive system diseases, inflammatory air way diseases, cellular immune inflammation diseases, hypersensitivities and allergies, vasular inflammation diseases, non-immune inflammatory disease, synovitis, villonodular synovitis, arthralgias, ankylosing spondylitis, spondyloarthritis, spondyloarthropathy, gout, Pagets disease, periarticular disorders such as bursitis, rheumatoid disease, rheumatoid arthritis and osteoarthritis, rheumatoid arthritis or osteoarthritis.
• an inflammatory disease such as inflammatory joint diseases, inflammatory connective tissue diseases, inflammatory autoimmune diseases, inflammatory myopathies, inflammatory digestive system diseases, inflammatory air way diseases, cellular immune inflammation diseases, hypersen
• Rheumatoid arthritis in particular, represents ongoing inflammation associated with severe pain.
• Arthritic pain is a form of inflammatory pain and arises from inflammation in a joint which causes both peripheral sensitization and central sensitization.
• the nociceptive system is activated by normally innocuous and nonpainful mechanical stimuli.
• pain is present and appears as spontaneous pain and hyperalgesia (augmented pain response on noxious stimulation and pain on normally nonpainful stimulation).
• Inflammatory processes in peripheral tissues lead to central sensitization in the spinal cord, which contributes to hyperalgesia and allodynia typically associated with inflammatory pain.
• Other types of inflammatory pain include inflammatory bowel diseases (IBD).
• Head pain including but not limited to migraine, migraine with aura, migraine without aura cluster headache, tension-type headache.
• Orofacial pain including but not limited to dental pain, temporomandibular myofascial pain, tinnitus, hot flushes, restless leg syndrome and blocking development of abuse potential.
• Further pain conditions may include, back pain, bursitis, dental pain, fibromyalgia or myofacial pain, menstrual pain, migraine, neuropathic pain (including painful diabetic neuropathy), pain associated with post-herpetic neuralgia, post-operative pain, referred pain, trigeminal neuralgia, visceral pain (including interstitial cystitis and IBS) and pain associated with AIDS, allodynia, burns, cancer, hyperalgesia, hypersensitisation, spinal trauma and/or degeneration and stroke.
• urogenital indications e.g. urinary incontinence, overactive bladder, emesis, cognitive disorders, anxiety, depression, sleeping disorders, eating disorders, movement disorders, glaucoma, psoriasis, multiple sclerosis, cerebrovascular disorders, brain injury, gastrointestinal disorders, hypertension, cardiovascular disease.
• the present invention relates to compounds of the Formula I:
• Ar 1 is phenyl optionally substituted by one or more substituents independently selected from OH, C 1-3 alkyl and halogen,
• Ar 1 is phenyl fused to a 5- or 6-membered heterocycle optionally substituted by one or more substituents independently selected from halogen, OH, and ⁇ O,
• Ar 2 is phenyl optionally substituted by one or more substituents independently selected from OH, C 1-3 alkyl and halogen,
• Ar 2 is phenyl fused to a 5- or 6-membered heterocycle optionally substituted by one or more substituents independently selected from halogen, OH, and ⁇ O,
• Gu is guanidinyl (or a tautomeric form thereof) optionally substituted by up to 4 substituents independently selected from C 1-3 alkyl,
• R 1 is independently H or C 1-5 alkyl optionally substituted by Y,
• Y is halogen, S(O) m —(C 1-3 alkyl), CO 2 —R 5 or Gu,
• R 5 is H or C 1-5 alkyl
• R 2 is H or C 1-3 alkyl
• R 1 and R 2 together with the carbon atom to which they are attached can be a cyclopropyl group
• R 3 and R 4 is H and the other is Q where Q is H, NH 2 , NHC(O)(C 1-6 alkyl), Gu, NHC(O)(CH 2 ) n NH2, NHC(O)(CH 2 ) n Gu, or (CH 2 ) n Gu,
• n 1, 2, 3, 4 or 5
• the invention is also directed to pharmaceutical compositions comprising a therapeutically effective amount of a compound of formula I herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
• the invention is also directed to a method of treating a disease or condition indicated for treatment with an opioid receptor agonist, particularly a ⁇ -opioid receptor agonist, in a subject, by administering to a subject in need thereof a therapeutically effective amount of one or more of the compounds herein, or a pharmaceutically acceptable salt thereof.
• an opioid receptor agonist particularly a ⁇ -opioid receptor agonist
• Ar 1 is phenyl optionally substituted by one or more substituents independently selected from OH and C 1-3 alkyl,
• Ar 1 is phenyl fused to a 5- or 6-membered heterocycle optionally substituted by ⁇ O.
• Ar 1 is 4-hydroxyphenyl, 2,6-dimethyl-4-hydroxyphenyl or benzoxazol-2-one-6-yl.
• Ar 1 is 2,6-dimethyl-4-hydroxyphenyl.
• Ar 2 is phenyl optionally substituted by one or more substituents independently selected from OH and C 1-3 alkyl.
• Ar 2 is 2,6-dimethyl-4-hydroxyphenyl or phenyl.
• Ar 2 is phenyl
• Gu is a guanidinyl optionally substituted by up to 4 methyl groups.
• Gu is guanidinyl or tetramethylguanidinyl.
• Gu is guanidinyl.
• R 1 is H or C 1-5 alkyl optionally substituted by Y, where Y is S(O) m —(C 1-3 alkyl) or CO 2 H.
• R 1 is C 1-5 alkyl optionally substituted by S(O)CH 3 or CO 2 H.
• R 1 is methyl, (CH 2 ) 5 CO 2 H or (CH 2 ) 2 S(O)CH 3 .
• R 1 is methyl
• R 2 is H.
• R 3 and R 4 is H and the other is Q where Q is H, NH 2 , NHC(O)CH 3 , guanidinyl, NHC(O)CH 2 NH 2 , NHC(O)CH 2 -guanidine, NHC(O)CH 2 CH 2 NH 2 , NHC(O)CH 2 CH 2 -guanidine or CH 2 -guanidine.
• R 3 is Q and R 4 is H.
• R 3 and R 4 are as follows:
• R 3 is guanidinyl and R 4 is H;
• R 3 is H and R 4 is H;
• R 3 is NHC(O)CH 3 and R 4 is H;
• R 3 is NH 2 and R 4 is H.
• R 3 is guanidinyl and R 4 is H.
• stereochemistry of the R 3 group is as shown in formula Ia below.
• Preferable groups of compounds of formula I, and Ia, and their pharmaceutically acceptable salts are those wherein:
• Ar 1 has the value of Ar 1 of any of the specific compounds mentioned below;
• Ar 2 has the value of Ar 2 of any of the specific compounds mentioned below;
• Gu has the value of Gu of any of the specific compounds mentioned below;
• R 1 has the value of R 1 of any of the specific compounds mentioned below;
• R 2 has the value of R 2 of any of the specific compounds mentioned below;
• R 3 has the value of R 3 of any of the specific compounds mentioned below; and/or
• R 4 has the value of R 4 of any of the specific compounds mentioned below.
• Preferred groups of compounds of formula I and Ia and their pharmaceutically acceptable salts are those wherein:
• Ar 1 is 4-hydroxyphenyl, 2,6-dimethyl-4-hydroxyphenyl or benzoxazol-2-one-6-yl;
• Ar 2 is 2,6-dimethyl-4-hydroxyphenyl or phenyl
• Gu is guanidinyl or tetramethylguanidinyl
• R 1 is methyl, (CH 2 ) 5 CO 2 H or (CH 2 ) 2 S(O)CH 3 ;
• R 2 is H
• R 3 and R 4 are as follows:
• R 3 is guanidinyl and R 4 is H;
• R 3 is H and R 4 is H;
• R 3 is NHC(O)CH 3 and R 4 is H;
• R 3 is NH 2 and R 4 is H.
• Halogen means a fluoro, chloro, bromo or iodo group
• Alkyl groups containing the requisite number of carbon atoms, can be unbranched or branched. Examples of alkyl include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl and t-butyl.
• “5- to 6-membered heterocycle” can include 1, 2 or 3 heteroatoms independently selected from N, O and S, and can be fully saturated, partially unsaturated, or fully unsaturated. Examples preferably include oxazole, or imidazole, or triazole, but can also be selected from pyrrole, furan, thiophene, pyrazole, isoxazole, thiazole, isothiazole, dithiazole, furazan, oxadiazole, thiadiazole, tetrazole, pyridine, pyran, thipyran, diazine, oxazine, thiazine, dioxin, triazine, tetrazine, and any fully saturated and partially unsaturated analogues thereof.
• “Pharmaceutically acceptable salts” of the compounds of formula I include the acid addition and base addition salts (including disalts, hemisalts, etc.) thereof.
• Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include the acetate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulphate/sulphate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, saccharate, stearate, succinate, tartrate, tosylate and trifluor
• Suitable base addition salts are formed from bases which form non-toxic salts. Examples include the aluminium, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts.
• the compounds of the invention include compounds of formula I and salts thereof as hereinbefore defined, polymorphs, and isomers thereof (including optical, geometric and tautomeric isomers) as hereinafter defined and isotopically-labelled compounds of formula I.
• compounds of formula (I) containing one or more asymmetric carbon atoms can exist as two or more stereoisomers.
• Cis/trans isomers may be separated by conventional techniques well known to those skilled in the art, for example, chromatography and fractional crystallisation.
• racemate (or a racemic precursor) may be reacted with a suitable optically active compound, for example, an alcohol, or, in the case where the compound of formula (I) contains an acidic or basic moiety, an acid or base such as tartaric acid or 1-phenylethylamine.
• a suitable optically active compound for example, an alcohol, or, in the case where the compound of formula (I) contains an acidic or basic moiety, an acid or base such as tartaric acid or 1-phenylethylamine.
• the resulting diastereomeric mixture may be separated by chromatography and/or fractional crystallization and one or both of the diastereoisomers converted to the corresponding pure enantiomer(s) by means well known to a skilled person.
• Chiral compounds of the invention may be obtained in enantiomerically-enriched form using chromatography, typically HPLC, on a resin with an asymmetric stationary phase and with a mobile phase consisting of a hydrocarbon, typically heptane or hexane, containing from 0 to 50% isopropanol, typically from 2 to 20%, and from 0 to 5% of an alkylamine, typically 0.1% diethylamine. Concentration of the eluate affords the enriched mixture.
• the present invention includes all pharmaceutically acceptable isotopically-labelled compounds of formula (I) wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature.
• isotopes suitable for inclusion in the compounds of the invention include isotopes of hydrogen, such as 2 H and 3 H, carbon, such as 11 C, 13 C and 14 C, chlorine, such as 38 Cl, fluorine, such as 18 F, iodine, such as 123 I and 125 I, nitrogen, such as 13 N and 15 N, oxygen, such as 15 O, 17 O and 18 O, phosphorus, such as 32 P, and sulphur, such as 35 S.
• isotopically-labelled compounds of formula (I), for example, those incorporating a radioactive isotope, are useful in drug and/or substrate tissue distribution studies.
• the radioactive isotopes tritium, i.e. 3 H, and carbon-14, i.e. 14 C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.
• substitution with heavier isotopes such as deuterium, i.e. 2 H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances.
• Isotopically-labelled compounds of formula (I) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples and Preparations using an appropriate isotopically-labelled reagents in place of the non-labelled reagent previously employed.
• Example a free amine compound of formula INT1 (below) can be converted to the corresponding compound of formula (I) by reaction with a carboxamidine such as 3,5-dimethylpyrazol-1-carboxamidine.
• a carboxamidine such as 3,5-dimethylpyrazol-1-carboxamidine.
• guanidine can be added simultaneously, or in sequence, or by combining fragments that each contain a guanidine in either a protected or unprotected form (an example of a protected product of this reaction is INT2 above (e.g. where “PG” is an appropriate protecting group such as Cbz—exemplified in the synthesis of Examples 1-9, or a Boc group—exemplified in the synthesis of Example 10, and relevant Preparations).
• PG is an appropriate protecting group such as Cbz—exemplified in the synthesis of Examples 1-9, or a Boc group—exemplified in the synthesis of Example 10, and relevant Preparations.
• Such intermediates can be constructed by assembling fragments through the formation of amide bonds from amines using standard bond coupling conditions such as those using mixed anhydrides or active esters of the relevant acid moiety.
• protecting groups may be applied, such as those described in standard texts such as “Protecting Groups” P. J. Kocie ⁇ ski, ed. Thieme; “Protective Groups in Organic Synthesis” Theodora W. Greene ed. Wiley; etc.
• tert-butoxycarbonyl (Boc) amines and guanidines may be used and subsequently deprotected under acidic conditions using, for example, hydrogen chloride or trifluoroacetic acid in an organic co-solvent such as dioxan or dichloromethane.
• benzyloxycarbonyl (Cbz) amines and guanidines may be used and subsequently deprotected under hydrogenolytic conditions using, for example, catalytic molecular hydrogenation or transfer hydrogenation protocols familiar to those skilled in the art.
• salts of a compound of formula (I) may be readily prepared by mixing together solutions of the compound of formula (I) and the desired acid or base, as appropriate.
• the salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent.
• the degree of ionisation in the salt may vary from completely ionised to almost non-ionised.
• the compounds of the invention intended for pharmaceutical use may be administered alone or in combination with one or more other compounds of the invention or in combination with one or more other drug agent (or as any combination thereof). Generally, they will be administered as a formulation in association with one or more pharmaceutically acceptable excipients.
• excipient is used herein to describe any biologically inactive ingredient other than the compounds and salts of the invention. The choice of excipient will to a large extent depend on factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.
• a compound of the formula I, or a pharmaceutically acceptable salt or solvate thereof, as defined above may be administered simultaneously (e.g. as a fixed dose combination), sequentially or separately in combination with one or more other drug agent.
• Exemplary additional agents could be selected from one or more of:
• compositions suitable for the delivery of compounds and salts of the present invention and methods for their preparation will be readily apparent to those skilled in the art. Such compositions and methods for their preparation may be found, for example, in ‘Remington's Pharmaceutical Sciences’, 19th Edition (Mack Publishing Company, 1995).
• Compounds and salts of the invention intended for pharmaceutical use may be prepared and administered as crystalline or amorphous products. They may be obtained, for example, as solid plugs, powders, or films by methods such as precipitation, crystallization, freeze drying, spray drying, or evaporative drying. Microwave or radio frequency drying may be used for this purpose.
• the compounds and salts of the invention may be administered directly into the blood stream, into muscle, or into an internal organ.
• Suitable means for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular and subcutaneous.
• Suitable devices for parenteral administration include needle (including microneedle) injectors, needle-free injectors and infusion techniques.
• Parenteral formulations are typically aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (preferably to a pH of from 3 to 9), but, for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water.
• excipients such as salts, carbohydrates and buffering agents (preferably to a pH of from 3 to 9)
• a suitable vehicle such as sterile, pyrogen-free water.
• parenteral formulations under sterile conditions may readily be accomplished using standard pharmaceutical techniques well known to those skilled in the art.
• solubility of compounds of formula (I) and salts used in the preparation of parenteral solutions may be increased by the use of appropriate formulation techniques, such as the incorporation of solubility-enhancing agents.
• Formulations for parenteral administration may be formulated to be immediate and/or modified release.
• compounds and salts of the invention may be formulated as a solid, semi-solid, or thixotropic liquid for administration as an implanted depot providing modified release of the active compound.
• An example of such formulations include drug-coated stents.
• the compounds and salts of the invention may also be administered topically to the skin or mucosa, that is, dermally or transdermally.
• Typical formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, dusting powders, dressings, foams, films, skin patches, wafers, implants, sponges, fibres, bandages and microemulsions. Liposomes may also be used.
• Typical carriers include alcohol, water, mineral oil, liquid petrolatum, white petrolatum, glycerin, polyethylene glycol and propylene glycol. Penetration enhancers may be incorporated [see, for example, Finnin and Morgan, J Pharm Sci, 88 (10), 955-958 (October 1999).]
• topical administration include delivery by electroporation, iontophoresis, phonophoresis, sonophoresis and microneedle or needle-free (e.g. PowderjectTM, BiojectTM, etc.) injection.
• the compounds and salts of the invention may also be administered intranasally or by inhalation, typically in the form of a dry powder (either alone, as a mixture, for example, in a dry blend with lactose, or as a mixed component particle, for example, mixed with phospholipids, such as phosphatidylcholine) from a dry powder inhaler or as an aerosol spray from a pressurised container, pump, spray, atomiser (preferably an atomiser using electrohydrodynamics to produce a fine mist), or nebuliser, with or without the use of a suitable propellant, such as 1,1,1,2-tetrafluoroethane or 1,1,1,2,3,3,3-heptafluoropropane.
• the powder may comprise a bioadhesive agent, for example, chitosan or cyclodextrin.
• a pressurised container, pump, spray, atomizer, or nebuliser may contain a solution or suspension of the compound(s) or salt(s) of the invention comprising, for example, ethanol, aqueous ethanol, or a suitable alternative agent for dispersing, solubilising, or extending release of the active, a propellant(s) as solvent and an optional surfactant, such as sorbitan trioleate, oleic acid, or an oligolactic acid.
• a solution or suspension of the compound(s) or salt(s) of the invention comprising, for example, ethanol, aqueous ethanol, or a suitable alternative agent for dispersing, solubilising, or extending release of the active, a propellant(s) as solvent and an optional surfactant, such as sorbitan trioleate, oleic acid, or an oligolactic acid.
• the drug product Prior to use in a dry powder or suspension formulation, the drug product is micronised to a size suitable for delivery by inhalation (typically less than 5 microns). This may be achieved by any appropriate comminuting method, such as spiral jet milling, fluid bed jet milling, supercritical fluid processing to form nanoparticles, high pressure homogenisation, or spray drying.
• comminuting method such as spiral jet milling, fluid bed jet milling, supercritical fluid processing to form nanoparticles, high pressure homogenisation, or spray drying.
• Capsules made, for example, from gelatin or HPMC
• blisters and cartridges for use in an inhaler or insufflator may be formulated to contain a powder mix of the compound or salt of the invention, a suitable powder base such as lactose or starch and a performance modifier such as l-leucine, mannitol, or magnesium stearate.
• the lactose may be anhydrous or in the form of the monohydrate, preferably the latter.
• Other suitable excipients include dextran, glucose, maltose, sorbitol, xylitol, fructose, sucrose and trehalose.
• a suitable solution formulation for use in an atomiser using electrohydrodynamics to produce a fine mist may contain from 1 ⁇ g to 20 mg of the compound or salt of the invention per actuation and the actuation volume may vary from 1 ⁇ l to 100 ⁇ l.
• a typical formulation may comprise a compound of formula (I) or salt thereof, propylene glycol, sterile water, ethanol and sodium chloride.
• Alternative solvents which may be used instead of propylene glycol include glycerol and polyethylene glycol.
• Suitable flavours such as menthol and levomenthol, or sweeteners, such as saccharin or saccharin sodium, may be added to those formulations of the invention intended for inhaled/intranasal administration.
• Formulations for inhaled/intranasal administration may be formulated to be immediate and/or modified release using, for example, poly(DL-lactic-coglycolic acid (PGLA).
• Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.
• the dosage unit is determined by a prefilled capsule, blister or pocket or by a system that utilises a gravimetrically fed dosing chamber .
• Units in accordance with the invention are typically arranged to administer a metered dose or “puff” containing from 1 to 5000 ⁇ g of the compound or salt.
• the overall daily dose will typically be in the range 1 ⁇ g to 20 mg which may be administered in a single dose or, more usually, as divided doses throughout the day.
• the compounds and salts of the invention may be administered rectally or vaginally, for example, in the form of a suppository, pessary, or enema.
• Cocoa butter is a traditional suppository base, but various well known alternatives may be used as appropriate.
• the compounds and salts of the invention may also be administered directly to the eye or ear, typically in the form of drops of a micronised suspension or solution in isotonic, pH-adjusted, sterile saline.
• Other formulations suitable for ocular and aural administration include ointments, biodegradable (e.g. absorbable gel sponges, collagen) and non-biodegradable (e.g. silicone) implants, wafers, lenses and particulate or vesicular systems, such as niosomes or liposomes.
• the compounds and salts of the invention may be combined with soluble macromolecular entities, such as cyclodextrin and suitable derivatives thereof or polyethylene glycol-containing polymers, in order to improve their solubility, dissolution rate, taste-masking, bioavailability and/or stability for use in any of the aforementioned modes of administration.
• soluble macromolecular entities such as cyclodextrin and suitable derivatives thereof or polyethylene glycol-containing polymers
• Drug-cyclodextrin complexes are found to be generally useful for most dosage forms and administration routes. Both inclusion and non-inclusion complexes may be used.
• the cyclodextrin may be used as an auxiliary additive, i.e. as a carrier, diluent, or solubiliser. Most commonly used for these purposes are alpha-, beta- and gamma-cyclodextrins, examples of which may be found in International Patent Applications Nos. WO 91/11172, WO 94/02518 and WO 98/55148.
• the total daily dose of the compounds and salts of the invention is typically in the range 0.1 mg to 200 mg depending, of course, on the mode of administration, preferred in the range 1 mg to 100 mg and more preferred in the range 1 mg to 50 mg.
• the total daily dose may be administered in single or divided doses.
• These dosages are based on an average human subject having a weight of about 65 kg to 70 kg. The physician will readily be able to determine doses for subjects whose weight falls outside this range, such as infants and the elderly.
• the dosage administered will, of course, vary with the compound or salt employed, the mode of administration, the treatment desired and the disorder indicated.
• the total daily dosage of the compound of formula (I)/salt/solvate (active ingredient) will, generally, be in the range from 1 mg to 1 gram, preferably 1 mg to 250 mg, more preferably 10 mg to 100 mg.
• the total daily dose may be administered in single or divided doses.
• the present invention also encompasses sustained release compositions.
• the pharmaceutical composition may, for example, be in a form suitable for parenteral injection as a sterile solution, suspension or emulsion, for topical administration as an ointment or cream or for rectal administration as a suppository.
• the pharmaceutical composition may be in unit dosage forms suitable for single administration of precise dosages.
• the pharmaceutical composition will include a conventional pharmaceutical carrier or excipient and a compound according to the invention as an active ingredient. In addition, it may include other medicinal or pharmaceutical agents, carriers, adjuvants, etc.
• Exemplary parenteral administration forms include solutions or suspensions of active compounds in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms can be suitably buffered, if desired.
• Suitable pharmaceutical carriers include inert diluents or fillers, water and various organic solvents.
• the pharmaceutical compositions may, if desired, contain additional ingredients such as flavorings, binders, excipients and the like.
• excipients such as citric acid
• disintegrants such as starch, alginic acid and certain complex silicates
• binding agents such as sucrose, gelatin and acacia.
• lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often useful for tableting purposes.
• Solid compositions of a similar type may also be employed in soft and hard filled gelatin capsules.
• Preferred materials include lactose or milk sugar and high molecular weight polyethylene glycols.
• the active compound therein may be combined with various sweetening or flavoring agents, coloring matters or dyes and, if desired, emulsifying agents or suspending agents, together with diluents such as water, ethanol, propylene glycol, glycerin, or combinations thereof.
• Dosage regimens may be adjusted to provide the optimum desired response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
• Dosage unit form refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
• the dose and dosing regimen is adjusted in accordance with methods well-known in the therapeutic arts. That is, the maximum tolerable dose can be readily established, and the effective amount providing a detectable therapeutic benefit to a patient may also be determined, as can the temporal requirements for administering each agent to provide a detectable therapeutic benefit to the patient. Accordingly, while certain dose and administration regimens are exemplified herein, these examples in no way limit the dose and administration regimen that may be provided to a patient in practicing the present invention.
• dosage values may vary with the type and severity of the condition to be alleviated, and may include single or multiple doses. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. For example, doses may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects such as toxic effects and/or laboratory values. Thus, the present invention encompasses intra-patient dose-escalation as determined by the skilled artisan. Determining appropriate dosages and regiments for administration of the chemotherapeutic agent are well-known in the relevant art and would be understood to be encompassed by the skilled artisan once provided the teachings disclosed herein.
• a pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses.
• a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
• the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
• parenteral dosages this may conveniently be prepared as a solution or as a dry powder requiring dissolution by a pharmacist, medical practitioner or the patient. It may be provided in a bottle or sterile syringe. For example it may be provided as a powder in a multicompartment syringe which allows the dry powder and solvent to be mixed just prior to administration (to aid long-term stability and storage). Syringes could be used which allow multiple doses to be administered from a single device.
• compositions of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered.
• the composition may comprise between 0.1% and 100% (w/w) active ingredient.
• a pharmaceutical composition of the invention may further comprise one or more additional pharmaceutically active agents.
• Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.
• parenteral administration of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue.
• Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like.
• parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal injection, and kidney dialytic infusion techniques.
• Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations as discussed below.
• Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents.
• the active ingredient is provided in dry (i.e. powder or granular) form for reconstitution with a suitable vehicle (e.g. sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.
• a composition of the present invention can be administered by a variety of methods known in the art.
• the route and/or mode of administration vary depending upon the desired results.
• the active compounds can be prepared with carriers that protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
• Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are described by e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, (1978).
• Pharmaceutical compositions are preferably manufactured under GMP conditions.
• compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution.
• This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein.
• Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example.
• Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides.
• compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.
• each active ingredient will vary depending upon any number of factors, including but not limited to, the type of animal and type of disease state being treated, the age of the animal, and the route(s) of administration.
• reaction times, number of equivalents of reagents and reaction temperatures may be modified for each specific reaction, and that it may nevertheless be necessary or desirable to employ different work-up or purification conditions.
• Examples 2-9 were similarly prepared according to the method described above for example 1, starting from the appropriate protected guanidine of formula 1.
• reaction mixture was then concentrated under reduced pressure and the residue was purified by column chromatography (silica, 9% methanol in dichloromethane as eluant increasing to DCM/MEOH/NH 3 80/20/2 as eluant) to afford the title compound as a colourless oil in 50% yield, 96 mg.
• the resulting solution was washed with a solution of citric acid followed by sodium bicarbonate solution.
• the organic phase was washed with brine then dried over MgSO 4 .
• the organics were filtered and evaporated leaving a small amount of a crude gum.
• the gum was purified using an ISCO chromatography system on a 12 g silica column using gradient elution from 95:5:0.5 to 80:20:5 DCM:MeOH:NH 3 to afford the title compound as a solid in 5% yield, 20 mg.
• the title compound was prepared from the product of Preparation 55 using the method of Example 10 on a scale of 400 mg in a yield of 64% following purification by reverse phase column chromatography (C-18, 26 g, 8 ml/minute, 5% to 15% CH 3 CN in water).
• Preparations 2-17 were similarly prepared according to the method described above for preparation 1, starting from the appropriate respective amine and acid as mentioned below.
• Preparations 19-32 were similarly prepared according to the method described above for preparation 18, starting from the appropriate boc protected amine.
• Preparations 34-38 were similarly prepared according to the method described above for preparation 33, starting from the appropriate cbz protected amine.
• N-(tert-butoxycarbonyl)-2,6-dimethyl-L-tyrosyl-N-[(2S)-2-amino-3-phenylpropyl]-D-alaninamide (330 mg, 0.64 mmol) from preparation 33 in THF (5 mL) was added N,N′-Bis(benzyloxycarbonyl)-1H-pyrazole-1-carboxamidine (268 mg, 0.71 mmol) and N,N-diisopropylethylamine (173 uL, 0.97 mmol). The reaction was stirred for 2 hours at 60° C. and then quenched by the addition of water (5 mL).
• Preparations 40-48 were similarly prepared according to the method described above for preparation 39, starting from the appropriate amine.
• 1,1,3,3-tetramethylurea (450 mg) was dissolved in anhydrous ether (5 mL) under N 2 .
• Oxayl chloride (390 uL) was added over 1 minute and the mixture was left to stir. After 5 minutes a solid had precipitated on the base of the flask, this was broken up and stirring was continued overnight. Volatiles were evaporated to give N-[chloro(dimethylamino)methylene]-N-methylmethanaminium chloride salt as a white solid.
• the title compound was prepared from the product of Preparation 57 (1.95 g) in MeOH (24 mL) to which was added ammonium formate (0.75 g, 12 mmol) and palladium hydroxide on carbon (20 w/w %, 83 mg, 0.12 mmol) at room temperature. The mixture was warmed to 60° C. for 3 hours, filtered through Arbocel, concentrated and used without further purification.
• the title compound was made following the method of preparation 2 using N ⁇ 5 ⁇ -[(benzyloxy)carbonyl]-N ⁇ 2 ⁇ -(tert-butoxycarbonyl)-D-ornithine (970 mg) and benzyl[(2S)-1-amino-3-phenylpropan-2-yl]carbamate (851 mg) to give a white powder (1.85 g) which was used without further purification.
• In vitro measurement of substrate metabolism can be determined using the microsomal cytochrome P450 mono-oxygenase system. This is a useful application in ranking clearance of a number of compounds by Phase I (including cytochrome P450) metabolism prior to in vivo assessment Measurement of intrinsic clearance using these methods also allows a comparison of species difference to be made to assist with translation of pharmacokinetic parameters from preclinical to clinical study.
• Human and rat Liver microsomal assays were performed using pooled microsomes from the Pfizer Global Supply (BD GentestTM). Chemical reagents were purchased from commercial sources (Sigma-Aldrich) and drug entities synthesised at Pfizer Global Research. Incubation mixtures contained 50 mM phosphate buffer pH 7.4, 5 mM MgCl 2 , 5 mM isocitric acid and 1 unit/ml isocitric dehydrogenase. The microsomes were defrosted at room temperature and sufficient volumes added to give a final concentration of 0.5 nmol cytochrome P450/ml. Following the addition of 1 ⁇ M of substrate, the incubation was pre-incubated at 37° C. for 5 minutes.
• the reaction was then initiated by the addition of 1 mM NADP and incubation aliquots are taken over a 1 h time course. The reaction was subsequently stopped by addition of acetonitrile cooled on ice. The incubation mixture was then centrifuged and the supernatant removed for injection onto LC-MS/MS system. Providing that the substrate concentration is below the Km, the metabolism should be first order giving a log-linear plot of substrate disappearance over time. The gradient of this line is the first order rate constant (k) and this maybe converted to estimate a substrates intrinsic clearance when factoring in the protein concentration.
• k the first order rate constant
• the intrinsic clearance in rat or human liver microsomes is calculated from:
• Mean ⁇ ⁇ LogD Mean ⁇ ⁇ log 10 ⁇ ( corrected ⁇ ⁇ peak ⁇ ⁇ area ⁇ ⁇ for ⁇ ⁇ octanol ⁇ ⁇ sample ) ( corrected ⁇ ⁇ peak ⁇ ⁇ area ⁇ ⁇ for ⁇ ⁇ buffer ⁇ ⁇ sample )
• the duplicate logD values must be with 0.4 log units of each other and the positive controls must be within 0.2 log units of the known logD value.

Priority Applications (2)

Applications Claiming Priority (2)

Related Child Applications (1)

Publications (1)

Family

ID=43778553

Family Applications (2)

Family Applications After (1)

Country Status (8)

Cited By (2)

Citations (1)

Family Cites Families (13)

• 2011
  • 2011-02-11 EP EP11707708A patent/EP2539358A1/fr not_active Withdrawn
  • 2011-02-11 CA CA2789606A patent/CA2789606A1/fr not_active Abandoned
  • 2011-02-11 WO PCT/IB2011/050580 patent/WO2011104649A1/fr active Application Filing
  • 2011-02-11 JP JP2012554454A patent/JP2013520489A/ja active Pending
  • 2011-02-24 UY UY0001033240A patent/UY33240A/es unknown
  • 2011-02-24 AR ARP110100560A patent/AR080297A1/es not_active Application Discontinuation
  • 2011-02-24 TW TW100106220A patent/TW201138763A/zh unknown
  • 2011-02-24 US US13/034,155 patent/US20110207654A1/en not_active Abandoned
• 2012
  • 2012-09-07 US US13/606,519 patent/US8598123B2/en not_active Expired - Fee Related

Patent Citations (1)

Non-Patent Citations (7)

Also Published As

Similar Documents

Legal Events