Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/18—Kallidins; Bradykinins; Related peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• This invention relates to compounds with combined bradykinin receptor antagonist and mu- opioid receptor agonist activities and to methods of using the same.
• C-Fiber afferents are known to mediate both the sensation of pain as well as the neurogenic
• neuropeptides include: substance-P, neurokinin A,
• neurokinin B calcitonin gene related peptide (CGRP), cholecystokinin (CCK), vasoactive intestinal polypeptide (VIP), and neuropeptide Y, among other neurotransmitters.
• CGRP calcitonin gene related peptide
• CCK cholecystokinin
• VIP vasoactive intestinal polypeptide
• neuropeptide Y neuropeptide Y
• molecular weight compounds such as morphine, oxymorphone, fentanyl and their derivatives will inhibit the release of the neuropeptides from peripheral C-fibers by acting as mu-opioid receptor agonists locally (at terminal mu-opioid receptors in the periphery) and in the CNS. This inhibition is independent of both the constellation of peptides contained in the specific C-fiber as well as the stimulus causing their release.
• bradykinin antagonist BKAn
• mu-opioid receptor agonist heterodimers These processes are bradykinin antagonist (BKAn)/mu-opioid receptor agonist heterodimers.
• BKAn mu-opioid receptor agonists are due to their ability to easily penetrate the CNS.
• BKAn mu-opioid receptor agonists are due to their ability to easily penetrate the CNS.
• heterodimers should not penetrate the CNS due to the highly cationic nature of the BKAn.
• mu-opioid receptor agonist activity should be limited to the periphery and
• the present invention provides heterodimeric compounds of the general formula
• BKAn bradykinin antagonist peptide
• Y is a mu-opioid receptor agonist
• X is a linking moiety. More specifically, the present invention provides heterodimeric compounds
• mu-opioid receptor agonist is selected from fentanyl, dihydromorphine and morphine or derivatives or analogs thereof.
• present invention also provides heterodimers comprising improved linking moieties as well as improved bradykinin antagonists.
• Figure 1 shows the effect of dihydromorphine (DHM) on paw licking time following formalin injection.
• Figure 2 shows the effect of CP-0840 on paw licking time following 10 ⁇ l formalin injection.
• Figure 3 shows the effect of dihydo ⁇ norphine on the response time of mice exposed to a hot surface.
• Figure 4 shows the effect of CP-0840 on the response time of mice exposed to a hot surface.
• Figure 5 compares the effect of saline, DHM, CP-0597 and CP-0840 on carrageenan (1%
• Figure 6 shows the duration of action of CP-0840 in rats.
• Figure 7 compares the effect of saline, DHM, CP-0597 and CP-0840 on mustard oil induced neurogenic inflammation in the rat hind paw.
• Figure 8 shows the effect of CP-0840 on the hypotensive response to bradykinin.
• Figure 9 shows the selectivity of CP-0840.
• the mu-opioid agonist is selected from fentanyl, dihydromorphine and morphine or derivatives or
• Y is
• R, and R 2 are independently selected from
• Y is
• Rl and R2 are independently selected from
• R is a linking group X of the formula CH 2 CH 2 (Phe)CH 2 C(O); R 4 is COCH 2 CH 3 .
• Preferred BKAn components include
• any of the above peptides may be substituted with L- Arg or L-Lys in the "0" position (i.e., D-Arg).
• the peptides may also be substituted with D- or L-Lys in the 0 to 6 positions for coupling to Y.
• Linkage may then be accomplished for example, via the ⁇ -amino group of the L-Arg residue, or the ⁇ -amino or e-amino group of the D-Lys or L- Lys residue.
• aqueous phase was acidified with IN aqueous hydrochloric acid and extracted with methylene
• dihydro-17-methylmorphinan was prepared as follows: a) 4,5 «-Epoxy-3-O-acetyl-6- «-hydroxy-7,8-didehydro-17-methylmorphinan:
• reaction mixture was transferred to a separatory funnel and extracted with chloroform (3x).
• didehydro-17-methylmorphinan was prepared as follows: a) 4,5- «-Epoxy-3-triphenylmethoxy-6- «-hydroxy-7,8-didehydro-17-methylmo ⁇ hinan:
• reaction mixture was diluted with methylene chloride and the organic phase separated.
• the aqueous phase was further extracted with methylene chloride.
• the combined organic layers were dried over magnesium sulfate. Filtration, removal of solvent, and column chromatography
• reaction mixture was diluted with ethylacetate and washed with brine, then water.
• Mass Spectral Analysis calculated (M+2) 1695; found (M+2) 1695.
• reaction mixture was diluted with methylene chloride and
• Example 4 The crude material was dissolved up into H 2 O/CH 3 CN/AcOH (8:2: 1) and purified by
• piperidinyl]propanamide was prepared as follows: a) 4-(4-carbomethoxymethylphenyl amine)- 1 -(2-phenethyl)piperidine: '
• reaction mixture was filtered through a plug of celite and
• piperidinyljpropanamide and CP-0597 was prepared by a similar coupling method as described in
• Example IV The crude heterodimer was dissolved into 10% AcOH/H 2 O and purified on RP-
• reaction mixture was poured into 80g of ice containing 93 ml of concentrated ammonium
• Example IV The crude heterodimer was purifed on RP-HPLC. Pure fraction were combined,
• piperidinyljpropanamide was prepared as follows: a) N-phenyl-3-(2-carbomethoxyethyl)-N-[ 1 -(2-phenethyl)-4-piperdinyl]propanamide
• the compounds were assayed for B2 receptor antagonist activity on guinea pig ileum,
• a cDNA library from human brain was obtained from Stratagene. The
• mu receptor sequence was selectively amplified from the cDNA library using nested PCR. The first
• chloride-purified pRc/CMV (Invitrogen) was also digested with Hind HI and Xba I using standard methodology. The products of the two digests were resolved on a 0.7% low melt agarose gel.
• Sections of gel containing the human mu receptor DNA (approximately 1.2 kb) and the pRc/CMV DNA (approximately 5.5 kb) were excised from the gel.
• the gel slices containing these DNAs were heated at 65 °C and aliquots combined in a reaction containing T4 DNA ligase. The reaction was incubated overnight at 15°C. An aliquot of this reaction was used to transform frozen competent E.
• nucleotide missinco ⁇ orations were detected and those that altered the amino acid sequence of the
• Cesium chloride-purified human mu receptor-pRc/CMV plasmid was transfected into CHO-K1 (ATCC) cells using the Lipofectamine reagent (GibcoBRL). Transfectants were
• hmu5 was chosen based upon binding levels, binding kinetics and inhibition patterns as the clone to
• Binding assays were performed by incubating human clone membrane solution (50ug/well in 125 ul final concentration) with 3 H-DAMGO (final concentration 5nM) with or without test compounds in assay buffer , at room temperature, for 60 minutes, at a final volume of 315 ul. All test compound
• RNA was isolated from human lung fibroblasts (CCD- 16 LU obtained from the ATCC) using
• the first round PCR used the two primers CTCCGAGGAGGGGTGGG
• PCR were done using the following conditions: 94°C, 1 minute for denaturation, 50°C, 1 minute for annealing followed by 72°C, 3 minutes for extension. Excess primers were removed with a Centricon
• pRc CMV (Invitrogen) was also digested with Hind III and Xba I using standard methodology. The
• CHO-Kl ATCQ cells using the Lipofectamine reagent (Gibco/BRL). Transfectants were selected
• Buffer A consisting of 25mM TES(pH 6.8)with 2uM 1,10-Phenanthroline, and centrifuged at 27,000xg for 15 min. this was then repeated.
• Buffer B Buffer A with 2uM Captopril,140ug/Ml Bacitracin, 0.1%BSA
• Binding assays were performed by incubating human clone membrane solution (Approx.
• Example XIX Mouse Formalin Test This test is a classical test for opiate and non-steroidal analgesic compounds. Mice are pretreated s.c. with vehicle or compound 30 minutes before injecting the formalin. lO ⁇ l of 5%
• Figures 1 and 2 show the effect of dihydromo ⁇ hine and the heterodimer, CP-0840, both of
• mice were placed on a surface maintained at 55°C and the
• reaction time was recorded at time intervals up to 240 minutes.
• the volume of the paw was measured before and after injection
• test compounds were injected s.c. 30 min before injecting the carrageenan.
• Carrageenan (1%) was
• Figure 5 compares the effect of pretreatment of the rats with saline, dihydromorhine, CP-0597
• CP-0840 (as does CP-0597) at this dose had a duration of action of greater than 6h in the rat against
• CP-0840 is showing a clear co-operativity phenomenon possibly reflecting an opiate sensitive component during the second
• Figure 7 compares the effect of dihydromo ⁇ hine, CP-0597 and CP-0840 in this model. At the doses used CP-0840 produced a significant inhibition of the edema response compared to saline controls.
• bradykinin 80pM. These were repeated in the presence of increasing dose infusions
• CP-0840 The dose of CP-0840 reducing the hypotensive response to bradykinin by 50% (ED50)
• bradykinin 80pM
• CP-0840 can be said to be a selective

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• Health & Medical Sciences (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Peptides Or Proteins (AREA)

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• 1996
  • 1996-06-04 EP EP96918098A patent/EP0832106A2/de not_active Withdrawn
  • 1996-06-04 AU AU60444/96A patent/AU6044496A/en not_active Abandoned
  • 1996-06-04 WO PCT/US1996/008923 patent/WO1996039425A2/en not_active Ceased
  • 1996-06-05 TW TW85106740A patent/TW407159B/zh not_active IP Right Cessation

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