WO2017199014A1 - Traitement de la tolérance aux opioïdes - Google Patents

Traitement de la tolérance aux opioïdes Download PDF

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Publication number
WO2017199014A1
WO2017199014A1 PCT/GB2017/051361 GB2017051361W WO2017199014A1 WO 2017199014 A1 WO2017199014 A1 WO 2017199014A1 GB 2017051361 W GB2017051361 W GB 2017051361W WO 2017199014 A1 WO2017199014 A1 WO 2017199014A1
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src
morphine
opioid
mice
tolerance
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PCT/GB2017/051361
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English (en)
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Tim HALES
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University Of Dundee
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • 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

Definitions

  • the present invention relates to the use of c-Src inhibitors for the mitigation of one or more side-effects associated with opioid analgesia, and in particular for the mitigation of opioid tolerance in human and veterinary medicine.
  • the present invention proposes the use of certain c-Src inhibitor compounds for the prevention or reduction or reversal of opioid tolerance.
  • Medicinal treatment of pain can be for the provision of immediate relief such as for example injury-related pain, or for post-surgery pain-relief as well as for provision of long- term relief for those suffering from chronic or persistent pain.
  • immediate relief such as for example injury-related pain
  • post-surgery pain-relief as well as for provision of long- term relief for those suffering from chronic or persistent pain.
  • World Health Organization pain ladder which provides guidelines for the selection of the type and level of analgesia i.e. medication which acts to relieve pain which is suitable for the initial treatment of different categories of pain.
  • Opioids have been used for thousands of years as analgesics and to this day are the recommended first treatment of choice on the three step WHO analgesic ladder recommended for the treatment of moderate and severe pain.
  • Opioid receptors and signalling pathways have evolved over millions of years to mediate endogenous pain control and reward in mammals. It has been documented that in addition to the desired pain control, this reward signal reinforces both beneficial behaviours such as feeding and reproduction as well as accounting for negative behaviours including opioid abuse and addiction.
  • the prototypical opioid agent, morphine is the gold standard to which all narcotic medications are compared and remains one of the most effective drugs available for treating severe pain. It is well known that there are adverse effects associated with the use of opioid-based drugs, both during the initial stages of drug-treatment (commencement of therapy), or where an existing therapy is amended i.e. the dose level or dose-frequency is altered (increased), or when a patient is rotated onto an alternative opioid analgesic.
  • opioids It is also well established that prolonged opioid treatment can lead to undesirable side effects/behaviours including constipation, immunosuppression, respiratory depression, hyperanalgesia, tolerance, physical dependency, and addiction. For these reasons it is recommended that the prescription of opioids is carefully monitored by health care providers.
  • Mu opioid receptors are responsible for the beneficial and detrimental effects of opioid drugs such as morphine. Mu receptors are uniquely distributed throughout the ascending and descending pain pathways and in brain regions involved in the affective component of pain. It is well established that prolonged treatment with opioid-based drugs, leads to opioid tolerance and a series of associated debilitating side-effects including respiratory depression, which is the primary cause of death during opioid overdose. There are increasing numbers of drug deaths linked to opioid use/misure in Scotland and elsewhere in the world Baldacchino A, et al. 2010. J Psychopharm 24: 1289; Agarin T, et al. 2015. Pain Physician. 18:E307). This underlines why the search for an opioid-based treatment for pain, which lacks the adverse effect of drug tolerance remains a key target for the global pharmaceutical industry.
  • the present invention provides c-Src inhibitors for use in mitigating side-effects associated with analgesic opioids, and in particular for the mitigation of opioid analgesic tolerance.
  • the present invention provides c-Src inhibitors for use in mitigating side-effects associated with analgesic opioids, and in particular mitigating opioid analgesic tolerance wherein the opioid analgesic tolerance is reduced in a subject undergoing opioid-based pain treatment or wherein the development of opioid analgesic tolerance is inhibited in a subject beginning opioid-based pain treatment.
  • the present invention provides c-Src inhibitors for use in mitigating side-effects associated with analgesic opioids, and in particular mitigating opioid analgesic tolerance, including reduction of opioid analgesic tolerance in a subject undergoing opioid-based pain treatment or inhibition of the development of opioid analgesic tolerance in a subject beginning opioid-based pain treatment, wherein the c- Src inhibitor is a potent c-Src inhibitor, or a potent and selective c-Src inhibitor, or a c-Src inhibitor with a K d for c-Src of between 0.1 and 0.5, preferably between 0.1 and 0.4, more preferably between 0.15 and 0.3 nM.
  • the present invention provides c-Src inhibitors for use in mitigating side-effects associated with analgesic opioids, and in particular mitigating opioid analgesic tolerance in accordance with any one, or combination of one or more of the aspects as detailed herein, wherein the c-Src inhibitor is a compound of general Formula I:
  • the present invention provides c-Src inhibitors for use in mitigating side-effects associated with analgesic opioids, and in particular mitigating opioid analgesic tolerance
  • the c-Src inhibitor is a compound of general Formula l(a) as defined hereinafter, and especially wherein the c-Src inhibitor is A/-(2-chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-1-piperazinyl]-2-methyl-4- pyrimidinyl]amino]-5-thiazole carboxamide monohydrate, also known as dasatinib or the free-amide or an alternative pharmaceutically acceptable salt thereof.
  • the present invention provides c-Src inhibitors for use in mitigating side-effects associated with analgesic opioids, and in particular treating opioid analgesic tolerance wherein the opioid analgesic tolerance is reversed in a subject undergoing opioid-based pain treatment and preferably wherein the c-Src inhibitor is a compound of Formula I, preferably Formula l(a), and especially wherein the c-Src inhibitor is dasatinib or the free-amide or an alternative pharmaceutically acceptable salt thereof.
  • the present invention provides c-Src inhibitors for use in mitigating side-effects associated with analgesic opioids, and in particular opioid analgesic tolerance in accordance with any one, or combination of one or more of the aspects as detailed herein, wherein the c-Src inhibitor is a compound of general Formula
  • the present invention provides c-Src inhibitors for use in mitigating side-effects associated with analgesic opioids, and in particular opioid analgesic tolerance wherein the c-Src inhibitor is a compound of general Formula I I, or a Group A compound of Formula II, and preferably is 4-amino-5-(4-chlorophenyl)-7- (dimethylethyl)pyrazolo[3,4-c pyrimidine, also known as (PP2) or a pharmaceutically acceptable salt thereof.
  • the present invention provides pharmaceutical compositions for use in accordance with any of the aspects as defined herein. According to another embodiment the present invention provides pharmaceutical compositions for use in the treatment of pain wherein the composition comprises the combination of at least one opioid analgesic and at least one c-Src inhibitor.
  • the present invention provides pharmaceutical compositions for use in the treatment of pain wherein the composition comprises the combination of at least one opioid analgesic and at least one c-Src inhibitor and wherein the composition is suitable for oral administration.
  • c-Src a new target protein in the pain pathway
  • c-Src which when inhibited, for the first time demonstrates dramatic reductions in opioid tolerance in a mouse model.
  • the Applicants have used mouse strains engineered to lack opioid receptors and/or components of opioid signalling pathways to confirm that this c-Src inhibition provides a similar level of attenuation of tolerance to that observed in mice lacking the -arrestin2 protein, known to be instrumental in morphine analgesic tolerance (Bohn L, Lefkowitz R, Gainetdinov R, Peppel K, Caron M, Lin F. 1999. Science 286: 2495).
  • the c-Src inhibitory-based approach developed by the Applicant has the potential for the selective stimulation of analgesia without the undesirable opioid tolerance associated with current drug therapies. As also discussed hereinafter this c-Src inhibitory-based approach has the potential to reduce undesirable side-effects of opioids, such as opioid tolerance, but without increasing their potential for addiction.
  • the present invention provides c-Src inhibitors for use in mitigating side- effects associated with analgesic opioids, and in particular mitigating opioid analgesic tolerance, and particularly c-Src inhibitors for use in reducing opioid analgesic tolerance in a subject undergoing opioid-based pain treatment or wherein the development of opioid analgesic tolerance is inhibited in a subject beginning opioid-based pain treatment.
  • the Applicants have demonstrated that the inhibition of a specific protein target, c-Src, reduces opioid tolerance in mice dosed with morphine.
  • the experimental results provided herein demonstrate that the c-Src inhibitors, dasatinib and PP2 inhibit the development of morphine analgesic tolerance in mice.
  • c-Src inhibitor dasatinib
  • this c-Src-inhibitor based approach would also reduce other undesirable side-effects of opioids without increasing their potential for addiction.
  • use of opioid agonists biased against c-Src activation may provide analgesia with fewer side effects.
  • the present invention provides c-Src inhibitors for use in mitigating side- effects associated with analgesic opioids including the mitigation of opioid analgesic tolerance.
  • the present invention provides c-Src inhibitors for use in mitigating one or more side-effects associated with analgesic opioids wherein the side-effects are one or more of opioid-induced drug tolerance, respiratory depression, constipation, immunosuppression and/or hyperalgesia.
  • the present invention additionally provides a combination of a c-Src inhibitor and an opioid-based analgesic for use in the treatment of pain.
  • c-Src INHIBITORS a combination of a c-Src inhibitor and an opioid-based analgesic for use in the treatment of pain.
  • Any suitable c-Src inhibitor may be used.
  • the identification of any known compound as a suitable c-Src inhibitor is considered to be within the ordinary skill of the skilled person.
  • the testing of whether any particular compound not previously known to be a c-SRC inhibitor can be accomplished using any one of the general methods as reported in the literature, and in particular methods detailed and referred to herein.
  • Exemplary c-Src inhibitors suitable for use herein are compounds of general Formula I and/or general Formula II as detailed herein.
  • the compounds of general Formula I are known inhibitors of protein tyrosine kinases, and especially as inhibitors of the Src- family kinases such as Lck, Fyn, Lyn, Src, Yes, Hck, Fgr and Blk.
  • Src- family kinases such as Lck, Fyn, Lyn, Src, Yes, Hck, Fgr and Blk.
  • dasatinib a compound of Formula I, has been reported by Montani et al.
  • the compounds of general Formula II are known inhibitors of tyrosine kinase, and in particular as Src-family tyrosine kinase inhibitors.
  • a compound of general Formula II is known as a selective c-Src kinase inhibitor. Brandvold et al. (Brandvold KR, Steffey ME, Fox CC, Soellner MB. Development of a highly selective c- Src kinase inhibitor. ACS chemical biology. 2012;7(8): 1393-1398.) refer to the selective c-Src kinase inhibitor, PP2, as previously reported by Hanke et al.
  • the present invention provides c-Src inhibitors for use in mitigating side-effects associated with analgesic opioids, and in particular mitigating opioid analgesic tolerance wherein the c-Src inhibitor is a compound of general Formula I:
  • Q is: a 5-membered heteroaryl ring; a 6-membered heteroaryl ring; or an aryl ring; optionally substituted with one or more R1 ;
  • R1 is : hydrogen or R6, where R6 is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, cycloalkenyl, cycloalkenylalkyl, aryl, aralkyl, heterocyclo, or heterocycloalkyl, each of which is unsubstituted or substituted with Z1 , Z2 and one or more (preferably, one or two) Z3, -OH, -OR6, -SH, -SR6 -C0 2 H, -C(0)qR6, or - 0-C(0)qR6 where q is 1 or 2, -S03H or -S(0)qR6, halo, cyano, nitro, -Z4-
  • R2 and R3 are each independently: hydrogen, R6, -Z4-R6, or -Z13-NR7R8 ;
  • R4 and R5 are each independently hydrogen or R6 ; are -Z4-N(R9)-Z5-NR10R 1 , -N (R9) Z4R6 ; or together with the nitrogen atom to which they are attached complete a 3- to 8- membered saturated or unsaturated heterocyclic ring which is unsubstituted or substituted with Zl, Z2 and Z3, which heterocyclic ring may optionally have fused to it a benzene ring itself unsubstituted or substituted with Zl, Z2 and Z3 ;
  • R7, R8, R9, R10, R1 1 and R12 are each independently hydrogen or R6;
  • R7 and R8 may together be alkylene, alkenylene or heteroalkyl, completing a 3-to 8-membered saturated or unsaturated ring with the nitrogen atom to which they are attached, which ring is unsubstituted or substituted with Z1 , Z2 and Z3 ; or any two of R9, R10 and R1 1 may together be alkylene or alkenylene completing a 3-to 8-membered saturated or unsaturated ring together with the nitrogen atoms to which they are attached, which ring is unsubstituted or substituted with Z1 , Z2 and Z3;
  • R13 is: cyano, nitro; -NH 2 , -NHOalkyl, -OH, -NHOaryl, -NHCOOalkyl, -NHCOOaryl,- NHS02alkyl, -NHS02aryl, aryl, heteroaryl, -Oalkyl, or -Oaryl ;
  • R14 is : nitro, -COOalkyl, or -COOaryl ;
  • R15 is: hydrogen, alkyl, aryl, arylalkyl, or cycloalkyl ;
  • Z1 , Z2 and Z3 are each independently: hydrogen or Z6, where Z6 is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, cycloalkenyl, cycloalkenylalkyl, aryl, aralkyl, alkylaryl, cycloalkylaryl, heterocyclo, or heterocycloalkyl ; a group (i) which is itself substituted by one or more of the same or different groups (i); or a group (i) or (ii) which is substituted by one or more of the following groups of the definition of Z1 , Z2 and Z3 ; -OH, -OZ6, - SH, -SZ6, -C(0)qH, -C(0)qZ6, -S0 3 H, -S03H,-S(0)qZ6, S(0)qN(Z9)Z6, halo, cyano, nitro, -Z4-NZ7Z8, -
  • Z4 and Z5 are each independently: a single bond, -Z11-S(0)q-Z12-, -Z11-C(0)-Z12-, - Z11-C(S)-Z12-, -Z11-0-Z12-, -Z11-S-Z12-, -Z11-0-C(0)-Z12-, or -Z11-C(0)-0-Z12-;
  • Z7, Z8, Z9 and Z10 are each independently hydrogen or Z6 ; or Z7 and Z8, or Z6 and Z10, may together be alkylene or alkenylene, completing a 3-to 8-membered saturated or unsaturated ring together with the atoms to which they are attached, which ring is unsubstituted or substituted with ZI, Z2 and Z3 ; or Z7 or Z8, together with Z9, may be alkylene or alkenylene completing a 3-to 8-membered saturated or unsaturated ring together with the nitrogen atoms to which they are attached, which
  • Z11 and Z12 are each independently; a single bond, alkylene, alkenylene, or alkynylene; and;
  • Z13 is; a single bond, -Z11-S(0)q-Z12-, -Z11-C(0)-Z12-, -Z11-C(S)-Z12-, -Z11-0-Z12-, - Z11-S-Z12-, -Z11-0-C(0)-Z12-, -Z11-C(0)-0-Z12-, -C(NR13)-, -C (CHR14)-, or -C (C (R14)2)-;
  • alk or "alkyl” refer to straight or branched chain hydrocarbon groups having 1 to 12 carbon atoms, preferably 1 to 8 carbon atoms.
  • the expression lower alkyl'Yefers to alkyl groups of 1 to 4 carbon atoms;
  • alkenyl refers to straight or branched chain hydrocarbon groups of 2 to 10, preferably 2 to 4, carbon atoms having at least one double bond. Where an alkenyl group is bonded to a nitrogen atom, it is preferred that such group not be bonded directly through a carbon bearing a double bond;
  • alkynyl refers to straight or branched chain hydrocarbon groups of 2 to 10, preferably 2 to 4, carbon atoms having at least one triple bond. Where an alkynyl group is bonded to a nitrogen atom, it is preferred that such group not be bonded directly through a carbon bearing a triple bond;
  • alkylene refers to a straight chain bridge of 1 to 5 carbon atoms connected by single bonds (e. g.,- (CH 2 ) x- wherein x is 1 to 5), which may be substituted with 1 to 3 lower alkyl groups;
  • alkenylene refers to a straight chain bridge of 2 to 5 carbon atoms having one or two double bonds that is connected by single bonds and may be substituted with 1 to 3 lower alkyl groups.
  • alkynylene refers to a straight chain bridge of 2 to 5 carbon atoms that has a triple bond therein, is connected by single bonds, and may be substituted with 1 to 3 lower alkyl groups.
  • aromatic cyclic groups for example 6 membered monocyclic, 10 membered bicyclic or 14 membered tricyclic ring systems
  • exemplary aryl groups include phenyl, naphthyl, biphenyl and anthracene;
  • cycloalkyl and “cycloalkenyl” refer to cyclic hydrocarbon groups of 3 to 12 carbon atoms;
  • halogen and halo refer to fluorine, chlorine, bromine and iodine;
  • unsaturated ring includes partially unsaturated and aromatic rings;
  • heterocycle refers to fully saturated or unsaturated, including non-aromatic (i. e.”heterocycloalkyl) and aromatic (i. e.”heteroaryl") cyclic groups, for example, 4 to 7 membered monocyclic, 7 to 11 membered bicyclic, or 10 to 15 membered tricyclic ring systems, which have at least one heteroatom in at least one carbon atom-containing ring.
  • Each ring of the heterocyclic group containing a heteroatom may have 1 ,2, 3 or 4 heteroatoms selected from nitrogen atoms, oxygen atoms and/or sulfur atoms, where the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quaternized.
  • the heterocyclic group may be attached at any heteroatom or carbon atom of the ring or ring system.
  • Exemplary monocyclic heterocyclic groups for compounds of Formula I include pyrrolidinyl, pyrrolyl, pyrazolyl, oxetanyl, pyrazolinyl, imidazolyl, imidazolinyl, imidazolidinyl, oxazolyl, oxazolidinyl, isoxazolinyl, isoxazolyl, thiazolyl, thiadiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, furyl, tetrahydrofuryl, thienyl, oxadiazolyl, piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolodinyl, 2- oxoazepinyl, azepinyl, 4-piperidonyl, pyridinyl, pyrazinyl, pyrimidiny
  • Exemplary bicyclic heterocyclic groups for compounds of Formula I include indolyl, benzothiazolyl, benzoxazolyl, benzodioxolyl, benzothienyl, quinuclidinyl, quinolinyl, tetra- hydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuryl, chromonyl, coumarinyl, benzopyranyl, cinnolinyl, quinoxalinyl, indazolyl, pyrrolopyridyl, furopyridinyl (such as furo [2, 3-c] pyridinyl, furo [3, 2-b] pyridinyl] or furo [2, 3-b] pyridinyl), dihydroisoindolyl, dihydroquinazolinyl (such as 3,4-dihydro-4-oxo-qui
  • Exemplary tricyclic heterocyclic groups for compounds of Formula I include carbazolyl, benzidolyl, phenanthrolinyl, acridinyl, phenanthridinyl, xanthenyl and the like, and the term “heteroaryl” refers to aromatic heterocyclic groups.
  • heteroaryl groups for compounds of Formula I include pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, isothiazolyl, furyl, thienyl, oxadiazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazolyl, triazinyl, and the like.
  • the present invention further provides c-Src inhibitors for use in mitigating side-effects associated with analgesic opioids, and in particular mitigating opioid analgesic tolerance wherein the c-Src inhibitor is a compound of general Formula 1(a) and salts thereof:
  • n 1 or 2; A is selected from carbon and nitrogen; B is selected from nitrogen, oxygen and sulfur; the five membered ring comprising A and B is aromatic; X3 is oxygen or sulfur; and Rl, R2, R3, R4 and R5 are as described above.
  • c-Src inhibitors for use in mitigating side-effects associated with analgesic opioids, and in particular mitigating opioid analgesic tolerance
  • the c-Src inhibitor is a compound of general Formula l(a)
  • c-Src inhibitors for use in mitigating side-effects associated with analgesic opioids, and in particular mitigating opioid analgesic tolerance wherein the opioid analgesic tolerance is reversed in a subject undergoing opioid-based pain treatment and preferably wherein the c-Src inhibitor is a compound of Formula I, preferably wherein the c-Src inhibitor is a compound of Formula l(a), and especially wherein the c-Src inhibitor is dasatinib or the free-amide or an alternative pharmaceutically acceptable salt thereof.
  • the present invention also provides c-Src inhibitors for use in mitigating side-effects associated with analgesic opioids, and in particular mitigating opioid analgesic tolerance wherein the c-Src inhibitor is a compound of general Formula II, and pharmaceutically-acceptable salts thereof wherein; X is C(R4) or N;
  • R1 is phenyl, mono-or di-halophenyl, mono-or di-alkoxy(C1 -C4)phenyl, mono- or di- alkyl(C1 -C4)phenyl, perhaloalkyl(C1 -C4)phenyl or nitrophenyl or said preceding R1 groups mono-substituted on alkyl(C1 -C4) or R1 (C1 -C6)alkyl is pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, furyl or thienyl;
  • R2 is phenyl, mono-or di-halophenyl, mono-or di-alkoxy(C1 -C4)phenyl, mono- or di- alkyl(C1 -C4)phenyl, perhaloalkyl(C1 -C4)phenyl or nitrophenyl or said preceding R2 groups mono-substituted on alkyl(C1-C4) or R2 is H, alkyl(C1-C6), cycloalkyl(C1-C7), pyridyl, halobenzoyl, alkoxy(C1-C4)benzoyl, alkyl(C1-C4)benzoyl, perhaloalkyl(C1- C4)benzoyl, nitrobenzoyl, naphthyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, fu
  • R3 is H, alkyl(C1-C4), morpholinoalkyl(C1-C4), carboxyalkyl(C1-C3) or alkoxy(C1- C4)carbonylalkyl(C1-C3);
  • R4 is phenyl, halophenyl, alkoxy(C1-C4)phenyl, alkyl(C1-C4)phenyl, perhaloalkyl(C1- C4)phenyl or said R, groups mono-substituted on alkyl(C1-C4) or R4 is cyano, H, halo, alkyl(C1-C6), alkoxy(C1-C4)carbonyl, alkanoyl(C1-C4), carbamoyl, or alkyl(C1- C4)carbamoyl.
  • halo is chloro, bromo, iodo, or fluoro and alkyl is a straight chain or branched saturated hydrocarbon.
  • the present invention provides c-Src inhibitors for use in mitigating side-effects associated with analgesic opioids, and in particular mitigating opioid analgesic tolerance wherein the c-Src inhibitor is a Group A compound of general Formula II wherein; R1 is alkyl(C1 -C4)phenyl or chlorophenyl; and
  • R2 is t-butyl or cyclohexyl; preferably wherein; R1 is chlorophenyl; and
  • R2 is cyclohexyl
  • R1 is chlorophenyl
  • R2 is t-butyl
  • R1 is 4-methylphenyl
  • R2 is cyclohexyl
  • R1 is 4-methylphenyl
  • R2 is t-butyl
  • c-Src inhibitors for use in mitigating side-effects associated with analgesic opioids, and in particular mitigating opioid analgesic tolerance
  • the c-Src inhibitor is a compound of general Formula II, is preferably a Group A compound, and in particular wherein the c-Src inhibitor is 4-amino-5-(4-chlorophenyl)-7- (dimethylethyl)pyrazolo[3,4-c ]pyrimidine, also known as PP2 or a pharmaceutically acceptable salt thereof.
  • the term "pharmaceutically acceptable salt” refers to those salts of the c- Src inhibitor compounds for use in accordance with the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable salts are well known in the art.
  • compositions of certain compounds of the formula (I) or formula (II) may be readily prepared either in situ during the final isolation and purification of c-Src compounds suitable for use in accordance with the invention, or they may be prepared in a conventional manner by mixing together solutions of a suitable compound, such as a compound of Formula I or II, and the desired acid, as appropriate.
  • a solution of a free base may be treated with the appropriate acid, either neat or in a suitable solvent, and the resulting salt isolated either by filtration or by evaporation under reduced pressure of the reaction solvent.
  • suitable salts see "Handbook of Pharmaceutical Salts: Properties Selection, and Use” by Stahl and Wermuth (Wiley- VCH, Weinheim, Germany, 2002).
  • non-toxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange.
  • acids for use as detailed herein include: fumarate, acetate, adipate, aspartate, alginate, ascorbate, benzoate, besylate (benzenesulfonate), bicarbonate/carbonate, bisulphate/sulphate, borate, butyrate, camphorate, camsylate (camphorsulfonate), citrate, cyclamate, cyclopentanepropionate , dodecylsulfate, edisylate, esylate (ethanesulfonate),, formate, fumarate, gluceptate, gluconate, digluconate, glucuronate, glucoheptonate, glycerophosphate, hexafluorophosphate, hemisulfate, heptanoate, hexanoate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iod
  • alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.
  • Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and aryl sulphonate.
  • c-Src inhibitory activity (IC 50 ) of any particular compound, including compounds of general Formula I or II, can be determined using the following method: 96-well assay plates are coated with enolase (100 ⁇ ) (Sigma Inc.) for 1 h at 37 °C. and then blocked with 300 ⁇ 0.5% BSA. A cell lysate containing the kinase of interest is prepared. The kinase of interest is produced by the baculovirus expression system in cells. The cells are lysed in 0.5% NP-40, 0.02M Tris, 150 mM NaCI and 1 % aprotinin.
  • the kinase is immunoprecipitated from the lysate with the appropriate antibody and subsequent incubation with protein-A coated Sepharose (Sigma, Inc., St. Louis, Mo.) beads.
  • the beads are washed four times in a 1 :10 bead to wash buffer volume ratio. They are resuspended to their final volume in kinase buffer and then aliquoted with an Eppendorf repeater pipet into appropriate assay wells.
  • Kininase buffer 25 mM HEPES, 3 mM MnC12, 0.1 mM Na3 V0 4 ).
  • the test compound (of Formula I or II) and gamma- 32P-ATP are then added to assay wells. After the final 20 min incubation, the assay wells are washed with an 1 mM ATP/50 mM EDTA buffer in two 9 sec wash cycles on a Microcell 96 Harvestor (Skatron Instruments, Sterling, Va.).
  • Scintillant is added to each well and the plate is read on a Micro-Beta Wallac, Inc. (Gaithersburg, Md.) reader. Generally, only alternate rows on an assay plate are used due to the inability of the crosstalk correction program of the Micro-Beta to correct for the high energy beta in adjacent wells. Samples are run in triplicate, averaged and then plotted to determine an IC 50 .
  • Opioid drugs act through mu, delta and kappa receptors; these are commonly referred to as MOPrs, DOPrs and KOPrs respectively, Dietis N, Rowbotham D, Lambert D. 2011. Br J Anaesth 107: 8. Opioid receptor activation inhibits adenylyl cyclase and voltage-activated Ca 2+ channels (VACCs) whilst activating inwardly rectifying K + channels.
  • VACCs voltage-activated Ca 2+ channels
  • MOPrs throughout the pain pathway mediate opioid analgesia.
  • MOPrs can combine with DOPrs to form MOPr/DOPr heteromers. Whilst not wishing to be bound to any particular theory, it has been proposed that MOPr/DOPr heteromers mediate the actions of analgesic opioids on nociceptive neurones (e.g. Walwyn W, John S, Maga M, Evans C, and Hales TG. 2009.
  • MOPrs are also located in the ventral tegmental area (VTA), a brain nucleus within the reward pathway that mediates the addictive properties of opioids (Johnson S, North R. 1992. J Neurosci 12: 483 and Matsui A, Williams J., 2011. J Neurosci 31 : 17729).
  • VTA ventral tegmental area
  • MOPrs are required for opioid analgesia. This is confirmed by the experimental results as discussed hereinafter and as illustrated in Figure 2A. Prolonged activation of MOPrs leads to tolerance, a process that involves a reduction in the availability of receptors on the cell surface through endocytosis (Williams J.T., et al. 2013. Pharmacol Rev 65: 223).
  • mice lacking one allele encoding the MOPr and referred to herein as heterozygous MOPr+/- mice, exhibit more rapid morphine tolerance than is seen in wild type (WT) mice containing both MOPr alleles.
  • WT wild type mice containing both MOPr alleles.
  • FIG 2B Heterozygous MOPr+/- mice lacking one allele have half the number of MOPrs (proteins) when compared to the strain of WT mice used in the experimental results discussed hereinafter (Matthes, H. W. et al. 1996. Nature 383: 819).
  • MOPr activation leads to phosphorylation by G protein coupled receptor (GPCR) kinases (GRKs) and recruitment of p-arrestin2 ( -arr2) triggering receptor endocytosis, a process that is instrumental in the development of opioid-drug induced tolerance.
  • GPCR G protein coupled receptor
  • -arr2-/- mice genetically engineered to lack -arr2 ( -arr2-/-) have been shown to develop negligible tolerance to opioid analgesia (Bohn L, Lefkowitz R, Gainetdinov R, Peppel K, Caron M, Lin F. 1999. Science 286: 2495). The Applicants have confirmed this observation in -arr2-/- mice. The results of these experiments are discussed hereinafter and are illustrated in Figure 3A.
  • -arr2-/- mice are also known to exhibit reduced respiratory depression and constipation (Raehal KM, Walker JK, Bohn LM. 2005. J Pharmacol Exp Ther. 314:1 195). As discussed hereinbefore, respiratory depression and constipation are also undesirable side-effects of opioid-based drug pain management therapies.
  • Desirable properties of c-Src inhibitors for use in accordance with the present invention include: high bioavailability and oral activity.
  • the tyrosine kinase system is a very complicated system that may provide multiple targets to modify the side effects of opioid drugs.
  • the present invention relates to the use of inhibitors of a particular member of the Src family of kinases, c-Src, for the mitigation of opioid analgesic tolerance.
  • Src family kinases in general are non-receptor tyrosine kinase inhibitors.
  • the Applicant has demonstrated a role for the tyrosine kinase c-Src, a member of the Src family of kinases, in the development of tolerance to the analgesic effects of morphine.
  • Src family kinase is a family of non-receptor tyrosine kinases with several members. These kinases are expressed at variable levels in different tissue types. There are 11 tyrosine kinases that are currently recognised to be part of the SFK family, including c-Src, Yes, Fyn, Fgr, Yrk, Lyn, Blk, Hck, and Lck. In the literature the nomenclature c-Src and Src are interchangeably used to refer to the c-Src tyrosine kinase within the SFK family.
  • an Src inhibitor as referred to herein means a compound which inhibits c-Src and optionally one or more members of the Src family kinase member.
  • c-Src inhibitor as referred to herein means a compound which is an inhibitor of c-Src, and is preferably a potent and/or potent and selective inhibitor of c-Src as defined hereinbefore.
  • Frk has homologs in invertebrates such as flies and worms, whilst Src homologs exist in organisms as diverse as unicellular choanoflagellates, The SrcA and SrcB subfamilies are specific to vertebrates
  • Src family members are not the same throughout all tissues and cell types. C-Src, Fyn and Yes are expressed ubiquitously in all cell types while the others are generally found in hematopoietic cells. Furthermore, of which c-Src, Fyn, Yes, Lck and Lyn are expressed at a high level in brain tissue (Keenan et al., 2015, FEBS Letters, 589, 1995-2000).
  • Src family kinases are structurally closely related and share the same regulatory domains.
  • Tyrosine kinases of Src family members contain the same typical structure: myristoylated terminus, a region of positively charged residues, a short region with low sequence homology, SH3 and SH2 domains, a tyrosine kinase domain, and a short carboxy-terminal tail.
  • SH3 and SH2 domains There are two important regulatory tyrosine phosphorylation sites. It is possible to repress kinase activity by phosphorylation of Tyr-527 in the carboxy-terminal tail of Src by the NRTK Csk.
  • v-Src an oncogenic variant of Src
  • This oncogenic v-Src is a product of the Rous sarcoma virus and as a result of an carboxy-terminal truncation, v-Src lacks the negative regulatory site Tyr-527 leading this enzyme to be constitutively active that in turn causes uncontrolled growth of infected cells.
  • substitution of this tyrosine with phenylalanine in c-Src results in activation.
  • a second regulatory phosphorylation site in Src is Tyr-416. This is an autophosphorylation site in the activation loop. It was found that a phosphorylation of Tyr-416 and Tyr-416 can suppressing the transforming ability of the activating Tyr-527 ⁇ Phe mutation by Tyr- 416 ⁇ Phe mutation leads to maximal stimulation of kinase activity.
  • Src family kinases were initially described in processes relating to cell proliferation and differentiation but they are now known to be widely expressed throughout the central nervous system in varying levels and involved in many different cellular processes (Salter and Kalia, 2004, Nature reviews. Neuroscience, 5,315-28). Neurones in particular express two different splice variants of c-Src that are known as N-Src (N1 and N2) as they have only been identified in neuronal cells (Keenan et al., 2015 as above).
  • c-Src has been identified in proximity to synaptic vesicles and it has been demonstrated that it will bind neuronal vesicular proteins including dynamin, a-adaptin and synapsin but does not directly phosphorylate these proteins suggesting that c-Src is involved in membrane trafficking in neuronal cells (Foster-Barber and Bishop, 1998, Proceedings of the National Academy of Sciences of the United States of America, 95, 4673-7). BAR2 not only mediates receptor desensitisation and internalisation but also the recruitment of c-Src following agonist binding.
  • BAR2 When BAR2 is bound to a GPCR it can provide a binding site for c-Src with the result that c-Src is part of a GPCR signalling complex with BAR2 (Luttrell and Luttrell, 2004 as above).
  • Dasatinib is a c-Src inhibitor, and it is used clinically to treat leukaemia, it has the ability to cross the blood brain barrier without modification (Lagas et al., 2009, Clinical cancer research : an official journal of the American Association for Cancer Research, 15, 2344-15, and Porkka et al., 2008, Blood, 1 12,1005-12). Dasatinib is a potent c-Src inhibitor that is normally administered orally to patients despite a low oral bioavailability.
  • tyrosine kinase inhibitor 3-(4-chlorophenyl) 1 -(1 ,1 - dimethylethyl)-1 H- pyrazolo[3,4-d]pyrimidin-4-amine (PP2), is a specific inhibitor of c-Src. As reported by Bain et al., 2007, The Biochemical Journal, 408, 297-315, and Uitdehaag ef al., 2012, British journal of pharmacology, 166, 858-76).
  • Imitanib can bind to c-Src but with a much lower affinity(K d >10 ⁇ ) compared to its other kinase targets (Seeliger ef al., 2007, Structure, 15, 299-31 1 ). However c-Src substrates have also been implicated in PDGFR signalling (Amanchy ef al., 2009, Mol Oncol, 3, 439-50).
  • PP2 is a selective inhibitor of c-Src that has been used predominately in vitro. It is unclear whether PP2 administered systemically crosses the BBB, however the benefit of using this drug is that it has an inactive analogue, PP3, that can be used as a matched control.
  • the second drug that we have used is dasatinib, this a clinically licensed drug that is used for the treatment of leukaemia. It has been extensively tested in vivo and does cross the blood brain barrier (BBB).
  • dasatinib is not a selective inhibitor of c-Src an also affects a number of other tyrosine kinases and their receptors including PDGFRp. As detailed hereinafter the Applicant have shown that dasatinib prevented the development of tolerance in WT mice. Furthermore, dasatinib, PP2 (but not PP3) has also been shown by the Applicant to inhibit the development of morphine tolerance in MOP+/- mice. MOP+/- mice are a good model to study tolerance development as they are significantly tolerant to the analgesic effects of morphine after 5 days of treatment. Dasatinib also reversed morphine tolerance in MOP+/- mice.
  • MOP receptors signal through BAR2 (perhaps with DOP receptors) to c-Src in which regulates the trafficking of GABAergic vesicles within the VTA.
  • dasatinib prevented the development of tolerance in WT mice. Furthermore, the Applicant has also now demonstrated that dasatinib, PP2 (but not PP3) inhibited the development of morphine tolerance in MOP+/- mice. The Applicant would propose that MOP+/- mice are a good model to study tolerance development because they have been shown to be significantly tolerant to the analgesic effects of morphine after 5 days of treatment. The Applicant has also shown that dasatinib also reversed morphine tolerance in MOP+/- mice.
  • the Applicant proposes that opioid receptors in the pain and reward pathways differ in their BAR mediated signalling mechanisms. To investigate this the Applicant has investigated whether the BAR2/c-Src system is differentially involved in opioid signalling in nociceptive pain and reward. To do this the Applicant has utilised mice that lack MOP receptors, DOP receptors and BAR2. We will also study the effects of morphine administration on the behaviour of a mouse that lacks both BAR2 and DOP receptors (BAR2-/-//DOP-/-). For all of these mouse models the Applicant has investigated the effects of these genetic manipulations on basal analgesia, analgesic tolerance and the development of morphine preference.
  • a c-Src inhibitor as defined herein means a compound which inhibits c-Src and optionally one or more members of the Src family kinase member.
  • the term c-Src inhibitor as referred to herein means a compound which is an inhibitor of c-Src, and is preferably a potent, or a potent and selective inhibitor of c-Src as defined hereinbefore.
  • Kd values can be readily determined using the methodology provided by Hulme, E. C. and Trevethick, M. A. (2010), Ligand binding assays at equilibrium: validation and interpretation. British Journal of Pharmacology, 161 : 1219-1237.
  • Drug tolerance as defined herein means, opioid induced analgesic tolerance.
  • Drug tolerance is the process by which a subject taking a drug for a prolonged period requires an increased amount of medication (dose) to provide an equivalent level of pain-relief as provided by the originally prescribed dose-level (starting dose) i.e. escalation of dose- level and/or dose-frequency over time.
  • Mitigation of a side-effect associated with opioid analgesia as defined herein means, reduction of, elimination of, prevention of, or delayed-on set of side-effects.
  • undesirable side-effects associated with analgesic opioids as defined herein include: opioid-induced drug tolerance; respiratory depression, constipation, immunosuppression; hyperalgesia and the like.
  • Mitigation of opioid analgesic tolerance in particular has long-been desirable in the field of chronic pain therapy because of the increased risks of the consequent adverse effects associated with prolonged opioid-based exposure.
  • the proposed use of certain c-Src inhibitors offers a unique possibility to realise the goal of mitigation of opioid analgesic tolerance, particularly reduction of, prevention of, elimination of, or delayed-on set of opioid analgesic tolerance.
  • the primary aim of the use of c-Src inhibitors for use in mitigating one or more side-effects associated with analgesic opioids, and particularly mitigation of opioid analgesic tolerance the Applicant has found that c-Src inhibitors may also be useful for the reversal of some side-effects associated with analgesic opiods.
  • c-Src inhibitor compounds can reverse pre-exisiting opioid analgesic tolerance. This is referred to herein as reversal of drug tolerance.
  • reversal of drug tolerance means that for a subject already taking a drug for a prolonged period and either experiencing or exhibiting signs of drug tolerance i.e. need for increased amounts of medication, the increased amount of medication can be dialled-back to the original dose-level (starting dose), either gradually or directly because the reason for the tolerance has been removed.
  • Physical dependency as defined herein means physiological adaptation to a substance such that the absence of the substance produces symptoms of withdrawal. Physical dependency is a product of tolerance.
  • Opioid agonists biased against c-Src means drugs that activate the MOPr causing G protein mediated signalling that result in analgesia, without activating c-Src mediated side effects.
  • the conventional assay for c-Src activation is measurement of phosphorylated c-Src (activated c-Src) using a phospho-c-Src specific antibody as referred to hereinafter.
  • An opioid drug as defined herein means any opioid analgesic drug. All opioids presently available for the treatment of pain are known to carry the risks of undesirable side-effects including drug-induced tolerance. Suitable opioid drugs for which opioid induced analgesic tolerance may be treated via use of the present c-Src inhibitory approach include: morphine, (5a,6a)-7,8-didehydro-4,5-epoxy-17-methylmorphinan-3,6-diol, and analogues and derivatives thereof, such as hydromorphine also known as dihydromorphine, 3,6-dihydroxy-(5a,6a)-4,5-epoxy-17-methylmorphinan, and diamorphine; codeine also known as 3-methylmorphine, (5a,6a)-7,8-didehydro-4,5- epoxy-3-methoxy-17-methylmorphinan-6-ol; dihydrocodeine, 4,5-a-epoxy-3-methoxy-17- methylmorphinan-6-ol; buprenorphin
  • compositions and formulations are provided.
  • the one or more c-Src inhibitor compounds or pharmaceutically acceptable salts thereof may be administered as the bulk substance, it is usually preferable to present the active ingredient in a pharmaceutical formulation, for example, wherein the agent is in admixture with at least one pharmaceutically acceptable carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
  • carrier refers to a diluent, excipient, and/or vehicle with which an active compound is administered.
  • the pharmaceutical compositions of the invention may contain combinations of more than one carrier.
  • Such pharmaceutical carriers can be sterile liquids, such as water, saline solutions, aqueous dextrose solutions, aqueous glycerol solutions, and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin, 18th Edition. The choice of pharmaceutical carrier can be selected with regard to the intended route of administration and standard pharmaceutical practice.
  • compositions may comprise, in addition to the carrier, any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), and/or solubilizing agent(s).
  • pharmaceutically acceptable refers to salts, molecular entities and other ingredients of compositions that are generally physiologically tolerable and do not typically produce untoward reactions when administered to a mammal (e.g., human).
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government for use in mammals, and more particularly in humans, or listed in the U.S. Pharmacopoeia or other generally recognized texts, for example the International Union of Pure and Applied Chemistry (lUPAC) Handbook of Pharmaceutical Salts, 2011 Edition.
  • a “pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes an excipient that is acceptable for veterinary use as well as human pharmaceutical use.
  • a “pharmaceutically acceptable excipient” as used in the present application includes both one and more than one such excipient.
  • the c-Src inhibitors for use in accordance with the present invention will normally, but not necessarily, be formulated into pharmaceutical compositions prior to administration to a patient. In one aspect, the invention is directed to use of a pharmaceutical composition comprising a c-Src inhibitor for the treatment of opioid tolerance.
  • the invention is directed to use of a pharmaceutical composition
  • a pharmaceutical composition comprising a c-Src inhibitor of Formula I or II, or a pharmaceutically acceptable salt thereof, and together with at least one or more pharmaceutically acceptable carrier.
  • the carrier must be "acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
  • the present invention further provides a pharmaceutical composition as defined herein for use as a medicament, for example for use as a medicament for use in the treatment of opioid tolerance.
  • the present invention is further related to use of a pharmaceutical composition comprising a c-Src inhibitor as defined hereinbefore for treatment of opioid tolerance.
  • compositions for use in accordance with the present invention may be in the form of oral, parenteral, transdermal, inhalation, sublingual, topical, implant, nasal, or enterally administered (or other mucosally administered) suspensions, capsules or tablets, which may be formulated in conventional manner using one or more pharmaceutically acceptable carriers or excipients.
  • the pharmaceutical composition is formulated for oral administration.
  • compositions for use in accordance with the invention include those in a form adapted for oral use in mammals including humans.
  • the pharmaceutical compositions for use in accordance with the invention include those in a form adapted for oral use and may be used for the treatment or mitigation of one or more side-effects associated with opioid analgesia and in particular for the treatment or mitigation of opioid tolerance, in mammals including humans.
  • compositions for use in accordance with the invention include one or more c-Src inhibitors as defined hereinbefore and can be administered for immediate-, delayed-, modified-, sustained-, pulsed- or controlled-release applications for example as a single or sole-therapeutic agent or may be administered as part of a combination therapy as detailed hereinafter.
  • the one or more c-Src inhibitors preferably compounds of Formula I or II, more preferably dasatinib and/or PP2, or pharmaceutically acceptable salts thereof, and further therapeutic agent(s), preferably one or more, opioid analgesic agents as detailed hereinafter may be employed in combination by administration simultaneously in a unitary pharmaceutical composition including both agents.
  • the combination may be administered separately in separate pharmaceutical compositions, each including one of the agents in a sequential manner wherein, for example, the c-Src inhibitor, preferably a compound of Formula I or II, more preferably dasatinib or PP2 or a pharmaceutically acceptable salt, thereof is administered first and the other agent, preferably an opioid analgesic second and vice versa.
  • the c-Src inhibitor preferably a compound of Formula I or II, more preferably dasatinib or PP2 or a pharmaceutically acceptable salt, thereof is administered first and the other agent, preferably an opioid analgesic second and vice versa.
  • Such sequential administration may be close in time (e.g. simultaneously) or remote in time.
  • administration of the other agent several minutes to several dozen minutes after the administration of the first agent, and administration of the other agent several hours to several days after the administration of the first agent are within the scope of the invention, wherein the lapse of time is not limited.
  • one agent may be administered once a day, and the other agent may be
  • the c-Src inhibitor as defined herein for use in accordance with the present invention is administered first.
  • the combination may be administered either in the same or different pharmaceutical composition.
  • the compound and agents When combined in the same formulation it will be appreciated that the compound and agents must be stable and compatible with each other and the other components of the formulation. When formulated separately they may be provided in any convenient formulation, conveniently in such manner as are known for such compounds in the art. During a treatment regime, it will be appreciated that administration of each agent of the combination may be repeated one or more times.
  • agents may be administered in the same or different dosage forms, e.g. one agent may be administered topically and the other compound may be administered orally. Suitably, both agents are administered orally.
  • kits or kit of parts
  • the combination kit can contain the agents in a single pharmaceutical composition, such as a tablet, or in separate pharmaceutical compositions.
  • the combination kit will contain each agent in separate pharmaceutical compositions either in a single package or in separate pharmaceutical compositions in separate packages.
  • the combination kit can also be provided with instructions, such as dosage and administration instructions.
  • dosage and administration instructions can be of the kind that are provided to a doctor, for example by a drug product label, or they can be of the kind that are provided by a doctor, such as instructions to a patient.
  • a c-Src inhibitor preferably a compound of Formula I or II, more preferably dasatinib or PP2, or a pharmaceutically acceptable salt thereof is used in combination with one or more additional therapeutic agents, preferably an opioid analgesic
  • the dose of the c-Src inhibitor compound or agent may differ from that when the compound or agent is used alone.
  • additional therapeutic agents preferably an opioid analgesic
  • the amount of any compound for use in accordance with the invention and the one or more additional therapeutic agents required for use in combination therapies or treatment as detailed herein will vary with the nature of the condition being treated and the age and the condition of the patient and will be ultimately at the discretion of the attendant physician or veterinarian.
  • the suitability of a potential combination of two or more compounds can be assessed on the basis of their in vitro or in vivo drug interactions.
  • the interactions of the two, or more, selected compounds are investigated in vitro using standard dose-response assays over a range of individualised concentrations. The selection of suitable conditions and concentrations for carrying out such investigations would be within the remit of the skilled practitioner.
  • compositions for use in the mitigation of side-effects associated with opioid analgesia which composition comprises one or more c-Src inhibitors as defined hereinbefore, preferably a compound of Formula I or Formula II, more preferably dasatinib or PP2, and one or more pharmaceutically acceptable, carriers, diluents or excipients.
  • compositions for use in the mitigation of side-effects associated with opioid analgesia which composition comprises one or more c-Src inhibitors as defined hereinbefore, preferably a compound of Formula (I) or Formula II, more preferably dasatinib or PP2, in combination with one or more opioids and one or more pharmaceutically acceptable, carriers, diluents or excipients.
  • c-Src inhibitors for use in mitigating one or more side-effects associated with analgesic opioids, preferably for mitigation of opioid analgesic tolerance
  • the c- Src inhibitor is dasatinib or PP2 and wherein the opioid is morphine.
  • composition comprising one or more c-Src inhibitors as detailed hereinbefore may be administered to an individual prior to, simultaneously, separately or sequentially with other therapeutic regiments or co-agents as desired.
  • the different actives may be formulated for the same or different delivery, for example one active formulated for immediate and another for sustained release. If a combined therapy is to be administered the active agents may be formulated for the same or different routes of administration as desired.
  • c-Src inhibitor compounds for use in accordance with the invention intended for pharmaceutical or veterinary or use may be 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.
  • c-Src inhibitors may be administered alone or in combination with one or more other c-Src inhibitors in accordance with the use of the present invention or in combination with one or more other therapeutic agents or drugs, and in particular one or more opioids as detailed hereinbefore (or as any combination thereof).
  • they will be administered as a formulation in association with one or more pharmaceutically or veterinarily acceptable excipients.
  • excipient' is used herein to describe any ingredient other than the c-Src compound(s). 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.
  • Pharmaceutically and veterinarily acceptable excipients include one or more of: lubricants, binding agents, diluents, surface-active agents, anti-oxidants, colorants, flavouring agents, preservatives, flavour enhancers, preservatives, salivary stimulating agents, cooling agents, co-solvents (including oils), emollients, bulking agents, anti- foaming agents, surfactants and taste-masking agents.
  • compositions suitable for use in the mitigation of one or more side-effects associated with opioid analgesia as detailed hereinbefore, 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 Gregory E. Hardee and J. Desmond Baggo, "Development and Formulation of Veterinary Dosage Forms", 2 nd Edition (CRC Press, 1998) and/or in Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995).
  • Formulations suitable for oral administration include solids, semi-solids or liquids such as tablets; soft or hard capsules; bolus; powders; lozenges (including liquid-filled); chews; multi and nano-particulates; gels; solid solutions; fast-dispersing dosage forms; fast- dissolving dosage forms; fast-disintegrating dosage forms; films; ovules; sprays; buccal/mucoadhesive patches; and liquid formulations.
  • Liquid formulations include suspensions, solutions, elixirs and syrups.
  • Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract, and/or buccal, lingual or sublingual administration by which the compound enters the blood stream directly from the mouth.
  • Liquid formulations may be employed as fillers in soft or hard capsules and typically comprise a carrier, for example, water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or a suitable oil, and one or more emulsifying agents and/or suspending agents. Liquid formulations may also be prepared by the reconstitution of a solid, for example, from a sachet.
  • a carrier for example, water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or a suitable oil
  • emulsifying agents and/or suspending agents may also be prepared by the reconstitution of a solid, for example, from a sachet.
  • Formulations for oral administration may be formulated to be immediate and/or modified release.
  • Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.
  • the formulation of tablets is discussed in "Pharmaceutical Dosage Forms: Tablets, Vol. 1", by H. Lieberman and L. Lachman, Marcel Dekker, N. Y., N.Y., 1980 (ISBN 0-8247-6918-X).
  • Tablets and capsules for oral administration may be in unit dose presentation form, and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricants, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants, for example potato starch; or acceptable wetting agents such as sodium lauryl sulfate.
  • the tablets may be coated according to methods well known in normal pharmaceutical practice.
  • Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use.
  • Such liquid preparations may contain conventional additives, such as suspending agents, for example sorbitol, methyl cellulose, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and, if desired, conventional flavouring or colouring agents.
  • suspending agents for example sorbitol, methyl cellulose, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agents, for example lecithin, sorbitan monooleate, or
  • c-Src inhibitor compounds or compositions comprising one or more c-Src compounds and optionally one or more opioid analgesics for use in the mitigation of one or more side-effects associated with opioid analgesia as detailed hereinbefore, may also be administered parenterally, or by injection 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, intrasynovial and subcutaneous.
  • a preferred parenteral administration route is intramuscular.
  • Suitable devices for parenteral administration include needle (including microneedle) injectors, needle-free injectors and infusion techniques.
  • compositions for use in the mitigation of one or more side-effects associated with opioid analgesia as detailed hereinbefore, wherein the composition is formulated for parenteral delivery comprising a c-Src inhibitor, particularly a compound of Formula I or II, or dasatinib and/or PP2, or a pharmacutically or veterinarily acceptable, salt thereof together with one or more pharmaceutically or veterinarily acceptable excipients.
  • the present invention further provides said composition formulated for parenteral delivery as an immediate release, or as a modified release formulation suitable for intramuscular or intravenous administration.
  • compositions for use in the mitigation of one or more side-effects associated with opioid analgesia as detailed hereinbefore, wherein the composition is formulated for parenteral or oral delivery comprising a c-Src inhibitor, particularly a compound of Formula I or II, or dasatinib and/or PP2, or a pharmacutically or veterinarily acceptable, salt thereof in combination with one or more opioid analgesics as defined hereinbefore, and preferably wherein the one or more opioid analgesics is morphine or a derivative or analogue thereof, together with one or more pharmaceutically or veterinarily acceptable excipients.
  • the present invention further provides said composition formulated for oral administration or parenteral delivery as an immediate release, or as a modified release formulation suitable for intramuscular or intravenous administration.
  • compositions for use in the mitigation of one or more side-effects associated with opioid analgesia as detailed hereinbefore, wherein the composition is formulated for oral adminstration comprising one or more c-Src inhibitors, particularly a compound of Formula I or II, or dasatinib and/or PP2, or a pharmacutically or veterinarily acceptable, salt thereof together with one or more pharmaceutically or veterinarily acceptable excipients.
  • the present invention further provides said composition formulated for parenteral delivery as an immediate release, or as a modified release formulation suitable for intramuscular or intravenous administration.
  • the present invention provides a pharmaceutical or veterinary composition for use in the mitigation of one or more side-effects associated with opioid analgesia as detailed hereinbefore, comprising a c-Src inhibitor, particularly a compound of Formula I or II, or dasatinib and/or PP2, or a pharmacutically or veterinarily acceptable, salt thereof, in combination with one or more opioid anagesics wherein the composition is formulated for immediate or modified release of the one or more c-Src inhibitors with delayed or later release of the one or more opioid analgesics.
  • a c-Src inhibitor particularly a compound of Formula I or II, or dasatinib and/or PP2
  • a pharmacutically or veterinarily acceptable, salt thereof in combination with one or more opioid anagesics
  • the composition is formulated for immediate or modified release of the one or more c-Src inhibitors with delayed or later release of the one or more opioid analgesics.
  • the present invention provides a pharmaceutical or veterinary composition formulated for oral, topical, intramuscular, rectal, subcutaneous or intravenous delivery comprising comprising one or more c-Src inhibitors, particularly a compound of Formula I or II, or dasatinib and/or PP2, or a pharmacutically or veterinarily acceptable, salt thereof together with one or more pharmaceutically or veterinarily acceptable excipients.
  • the present invention further provides said composition formulated for delivery as an immediate release, slow release or as a modified release formulation.
  • compositions for use in accordance with the invention may also be administered topically, (intra )dermally, or transdermally to the skin or mucosa.
  • 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.
  • compositions for use in accordance with 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 alternatives may be used as appropriate.
  • compositions for use in accordance with the present invention may be formulated to be immediate and/or modified release.
  • Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.
  • a physician will determine the actual dosage which will be most suitable for an individual human subject, or a veterinarian for an individual mammal (animal).
  • the specific dose level and frequency of dosage for any particular human or animal may be varied and will depend upon a variety of factors including the condition being treated, the inhibitory activity of the specific c-Src inhibitor employed and the re-exisiting or proposed opioid analgesic treatment to be/being employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the humand or animal to be treated.
  • a therapeutically effective amount of any c-Src inhibitor compound for use in accordance with the present invention can be determined by methods known in the art. As indicated hereinbefore the therapeutically effective quantities will depend on the age and on the general physiological condition of the subject, the route of administration and the pharmaceutical formulation used.
  • the therapeutic doses will generally be between about 1 and 2000 mg/day, for example between about 500 mg and 2000 mg/day.
  • the daily dose as employed for human treatment will range from 1 to 2000 mg, which may be administered in one or more daily doses, for example, depending on the route of administration and the condition of the subject.
  • each unit will contain 1 mg to 2000 mg of active ingredient.
  • the dosage form is a tablet, the total weight of the tablet is suitably 1000mg or lower.
  • c-Src compounds should be assessed for their biopharmaceutical properties, such as for example, solubility, solution stability (across a range of pHs), likely dose level and permeability.
  • solubility such as for example, solubility, solution stability (across a range of pHs), likely dose level and permeability.
  • PP2 and dasatinib as detailed herein have been used in clinical trials and/or have been used commercially for purposes other than mitigation of side-effects associated with opioid analgesia.
  • the desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example as one, two, three, four or more doses per day. If the compounds are administered transdermal ⁇ or in extended release form the compounds could be dosed once a day or less.
  • the compound is conveniently administered in unit dosage form; for example, containing 0.01 to 50 mg/kg of active ingredient.
  • These dosages are based on an average subject having a weight of about 20kg to 200Kg, and more particularly 50kg to 100kg.
  • the physician or veterinarian or livestock owner will readily be able to determine doses for humans or animals whose weight falls outside this range.
  • two or more pharmaceutical or veterinary compositions at least one of which contains a c-Src inhibitor for use in accordance with the present invention, may conveniently be combined in the form of a kit suitable for co-administration of the compositions.
  • An exemplary kit for use in accordance with the present invention comprises two or more separate pharmaceutical or veterinary compositions, at least one of which contains a c- Src inhibitor for use in accordance with the present invention, preferably a c-Src inhibitor of Formula I or Formula II, more preferably dasatinib or PP2, and suitable means for separately retaining said compositions, such as a container, divided bottle, or divided foil packet.
  • a c- Src inhibitor for use in accordance with the present invention preferably a c-Src inhibitor of Formula I or Formula II, more preferably dasatinib or PP2
  • suitable means for separately retaining said compositions such as a container, divided bottle, or divided foil packet.
  • a preferred exemplary kit for use in accordance with the present invention comprises two or more separate pharmaceutical or veterinary compositions, one of which contains a c- Src inhibitor for use in accordance with the present invention, preferably a c-Src inhibitor of Formula I or Formula II, more preferably dasatinib or PP2, and another separate pharmaceutrical or veterinary composition containing one or more opioid analgesics, as defined hereinbefore, and preferably morphine or a derivative or analogue thereof, and suitable means for separately retaining said compositions, such as a container, divided bottle, or divided foil packet.
  • a c- Src inhibitor for use in accordance with the present invention preferably a c-Src inhibitor of Formula I or Formula II, more preferably dasatinib or PP2
  • another separate pharmaceutrical or veterinary composition containing one or more opioid analgesics, as defined hereinbefore, and preferably morphine or a derivative or analogue thereof, and suitable means for separately
  • kits for use in accordance with the present invention are the familiar blister pack used for the packaging of tablets, capsules and the like.
  • Another example of a kit suitable for use in accordance with the present invention is a plurality of vials or other container, wherein one group of one or more vials contains a liquid formulation comprising a specific dosage of the c-Src inhibitor, as defined hereinbefore, and another group of one or more vials contains a liquid formulation comprising a specific dosing of an opioid analgesic such that the liquid formulation in each vial may be ready for direct injection into a human or animal.
  • one or more inhibitors of c-Src preferably one or more c- Src inhibitors of Formula I or II, more preferably dasatinib or PP2 can be administered simultaneously or concurrently with one or more opioids, preferably one or more of the opioid as detailed hereinbefore, and in particular with morphine or an analogue or derivative thereof for the provision of effective analgesia with mitigation of one or more side effects associated with opioid analgesia and in particular the inhibition of drug tolerance.
  • the c-Src and opioids are provided either in a kit format wherein the c-Src is to be administered prior to the opioid, or wherein the c-Src inhibitor and opioids are provided in a combined dosage form wherein the c-Src inhibitor containing-portion of the dosage form is formulated for immediate release and wherein the opioid inhibitor-containing portion of the dosage form is formulated for modified or delayed release.
  • dasatinib or PP2 can be administered simultaneously or concurrently with one or more opioids, preferably one or more of the opioid as detailed hereinbefore, and in particular with morphine or an analogue or derivative thereof for the provision of effective analgesia with mitigation of one or more side effects associated with opioid analgesia and in particular the inhibition of drug tolerance.
  • the dasatinib or PP2 and morphine or an analogue or derivative thereof are provided either in a kit format wherein the dasatinib or PP2 is to be administered prior to the morphine or an analogue or derivative thereof, or wherein the c- Src inhibitor and morphine or an analogue or derivative thereof are provided in a combined dosage form wherein the dasatinib or PP2-containing portion of the dosage form is formulated for immediate release and wherein the morphine or an analogue or derivative thereof-containing portion of the dosage form is formulated for modified or delayed release.
  • references to mitigation of one or more side-effects associated with analgesic opioids as used herein includes inhibition, or reduction as well as treatment via the alleviation of established symptoms of a condition (side-effect) i.e. including prevention of progression or control.
  • mitigation means treatment of a side-effect wherein "treat”, “treating” or “treatment” in reference to a disease, state, disorder, side- effect or condition includes: (1 ) to ameliorate the disease or one or more of the biological manifestations of the disease via (2) to interfere with (a) one or more points in the biological cascade that leads to or is responsible for the disease or (b) one or more of the biological manifestations of the disease, (3) to alleviate one or more of the symptoms or effects associated with the disease, (4) to slow the progression of the disease or one or more of the biological manifestations of the disease, (5) to diminish the likelihood of severity of a disease or biological manifestations of the disease, (6) delaying the appearance of clinical symptoms of the disease, state, disorder or condition developing in a mammal that may be afflicted with or predisposed to the disease, state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition, (7) inhibiting the disease, state, disorder or condition, i.e
  • a pharmaceutical composition comprising a c-Src inhibitor, or c-Src compounds, or compositions containing the c-Src inhibitors, or c-Src compounds optionally in combination with an opioid, together with one or more pharmaceutically acceptable, carrier, diluent or excipient; a composition comprising an c-Src inhibitor in combination with an opioid, for use as a medicament; a c-Src inhibitor, or a composition containing an c-Src inhibitor optionally in combination with an opioid for use in the mitigation of one or more side-effects associated with analgesic opioids
  • a veterinary composition comprising a c-Src inhibitor in combination with an opioid together with one or more acceptable carrier, diluent or excipient.
  • Figure 1 illustrates the reduction in morphine-induced analgesic tolerance by measurement of MPE over time (days) via inhibition of c-Src in different mouse types when treated with dasatinib (Drug X) or PP2 (Drug Y) and the reversal of morphine- induced analgesic tolerance in mice when dasatinib was administered 30 minutes prior to morphine.
  • Figure 1A illustrates the results obtained using dasatinib and wild type C57BL/6 (WT) mice
  • Figure 1 B illustrates the results obtained using dasatinib in MOPr+/- mice
  • Figure 1 C illustrates the results obtained using PP2 in MOPr+/- mice
  • Figure 1 D illustrates the results obtained using dasatinib when administered 30 minutes prior to morphine in in MOPr+/- mice.
  • Figure 2 illustrates morphine analgesia in WT and MOPr+/- mice which was assayed by measuring the latency for tail withdrawal from heated water.
  • Figure 3 illustrates the relative levels of morphine analgesic tolerance of different mouse genotypes. Detailed description of Figures
  • Figure 1 The results illustrated in Figures 1A to 1 D illustrate that the inhibition of c-Src by either dasatinib (Drug X) or PP2 (Drug Y) reduces morphine analgesic tolerance are provided in graph-format.
  • the results for the Vehicle confirmed that wild type C57BL/6 (WT) mice rapidly developed tolerance to morphine-induced analgesia when dosed with 10mg/Kg morphine subcutaneously (sc) once daily.
  • sc subcutaneously
  • FIG. 1 B illustrates the results obtained where MOPr+/- mice were subjected to the same treatments as for Figure 1A.
  • Figure 1 B the tolerance to morphine (10mg/Kg, once daily, sc) developed faster and became more marked in MOPr+/- mice receiving vehicle, however, MOPr+/- mice receiving an ip injection of dasatinib 30 mins prior to morphine injection exhibited negligible tolerance.
  • Figure 1 C illustrates the results obtained when MOPr+/- mice were dosed with PP2 and an inactive analogue thereof PP3 at 5mg/Kg, ip, 30 minutes prior to a daily injection of morphine (l Omg/Kg, sc). In the mice dosed with PP2 the tolerance to morphine-induced analgesia was significantly reduced whilst the mice dosed with the analogue rapidly developed tolerance.
  • MPE represents maximal possible effect for prolongation of tail withdrawal latency which is limited to 15 seconds (s) to prevent tail damage.
  • Statistics Data are mean ⁇ SEM, statistical significance was determined by one way repeated measures ANOVA with post hoc Tukey's test; *p ⁇ 0.05, **p ⁇ 0.01 , ***p ⁇ 0.001 .
  • Figure 2 In Figure 2 the results obtained when morphine analgesia in WT and MOPr+/- mice was assayed via measurement of the latency for tail withdrawal from water heated to 52°C are shown in graph-format.
  • Figure 3 In Figure 3 the results obtained when morphine analgesic tolerance and cause conditioned place preference (CPP) potency were assessed for different mouse varieties are shown in graph-format. In general mice lacking -arr2 are shown to exhibit reduced morphine analgesic tolerance and more potent CPP. In Figure 3A mice lacking one allele ( ⁇ -arr 2+I-) and mice lacking both alleles ( -arr2-/-) of the ⁇ -allele 2 are shown to exhibit a significant reduction in analgesic tolerance to morphine when compared to WT mice. All the mice in the experiments illustrated in Figure 3A were dosed once daily with morphine (10mg/Kg, sc).
  • dasatinib (commercially available from Bristol-Myers Squibb) was dissolved in DMSO (Sigma-Aldrich) to give a 50 mg/ml stock.
  • the final concentration of the dasatinib solution for injection was 1 mg/ml dasatinib to allow administration of 5 mg/Kg.
  • Kolliphor® EL a polyoxyl 35 hydrogenated castor oil, also known as a PEG-35 castor oil available from Sigma-Aldrich
  • PP2 was also dissolved in DMSO at its solubility limit of 25 mM and diluted in a 0.9% saline solution to give a final concentration of 1 mg/ml PP2 (16% DMSO and 16% Kolliphor EL).
  • PP3 (1 -phenyl-1 H-pyrazolo[3,4-d]pyrimidin-4-amine) has a higher solubility limit than dasatinib or PP2 and was made up in DMSO at 100 mM and diluted in a 0.9% saline solution to give a final concentration of 1 mg/ml of PP3 (5% DMSO and 5% Kolliphor® EL). These drugs were all administered via the intraperitoneal (IP) route. For the experiments as detailed herein, the matched vehicle injections contained all the constituents of the compound-containing injections with the exception of the active drug compound.
  • Opioid analgesia Opioid analgesia:
  • MOP-/- mice were first generated by Dr Brigitte Kieffer's lab in 1996 (Matthes et a/., 1996). They disrupted the Oprml gene utilising a technique called homologous recombination. This work involved the insertion of a neomycin resistant cassette into Exon 2 of the gene and was performed on embryonic stem cells from the 129/Sv mouse line, also known as the 129S1 mouse line, which is commercially available from The Jackson Laboratory (JAX), Connecticut, USA.
  • mice are now commercially available fully backcrossed onto C57BL/6J background from The Jackson Laboratory (JAX), Connecticut, USA, under stock number 007559.
  • the DOP-/- mice used herein were first generated by Dr Brigitte Kieffer's lab. They inactivated the Oprdl gene by targeting Exon 1 for deletion with a neomycin cassette. This was inserted into embryonic stem cells from the 129/Sv mouse line and further breeding was performed with C57BL/6J mice (Filliol et al., 2000). The mice are now commercially available fully backcrossed onto C57BL/6J background from The Jackson Laboratory (JAX) (stock number 007557). The BAR2-/- mice were initially developed by Dr Robert Lefkowitz's lab in 1999.
  • mice were created by utilising a homologous recombination technique targeting Exon 2 of the ⁇ -arrestin 2 gene on Chromosome 1 1 . These mice were again created using embryonic stem cells from the mouse line 129/Sv and further breeding and backcrossing performed on the C57BL/6J background (Bohn et al., 1999). These mice are also commercially available from The Jackson Laboratory (JAX), under stock number 01 1 130.
  • the MOP-/-, DOP-/- and BAR2-/- mice used in the experiments detailed herein were provided by Dr Wendy Walwyn at UCLA, these mice were maintained on a C57BL/6J background. All mice were maintained in the Medical Resource Unit of the University of Dundee in accordance with Home Office regulations.
  • mice had free access to food and water with 12 hour cycles of light and dark corresponding to day/ night externally. All of the experiments detailed herein were performed in the light phase. All work was performed by trained personnel in accordance with a UK Government Home Office project licence (Hales). For three days prior to each experiment mice were handled and habituated to the room where the tests were to take place. The room temperature was maintained between 19 and 21 °C. All experiments took place during daytime hours. All drug doses were calculated using individual body weight, maximum volume administered in a single injection was 200 ⁇ _. Groups of mice were made up of equal numbers of males and females where possible and balanced numbers when not. Mice were aged from 7 weeks to 24 weeks of age at the time of their participation in the tasks.
  • Genotyping was initially performed in-house using the following standard protocols but subsequently contracted to Transnetyx Inc., TN, USA.
  • Genomic DNA was extracted from ear clippings and sequenced using standard protocols. The protocol used is as follows. Extraction solution, commercially available from Sigma-Aldrich, 50 ⁇ _ was added to the ear clip sample along with 12.5 ⁇ _ of tissue preparation solution, also available from Sigma-Aldrich. These were allowed to incubate at room temperature for 10 minutes, then at 95°C on the heat block for a further 3 minutes. Neutralisation solution (50 ⁇ _, Sigma-Aldrich) was then added to stop the reaction. The resulting genomic DNA was either used immediately for PCR or stored at - 20°C until required.
  • a master-mix was created for each PCR reaction as required (Table 1.1 ) and the samples set up on the thermo-cycler using the appropriate programme.
  • the primers used for each reaction consisted of a forward, reverse and middle primer, the sequences of which can be viewed in Table 1 .2, they were obtained from Sigma-Aldrich. Following completion of the PCR reactions the samples were maintained at 4°C until an electrophoresis gel could be run.
  • Table 1 .1 shows the PCR master-mix recipe use for each of the mouse genotypes. This was varied between the genotypes as detailed above.
  • the primer sequences for each reaction can be viewed in Table 1 .2.
  • the reagents were supplied by Sigma-Aldritch and Fisher Scientific.
  • Table 1 .2 shows the forward, reverse and middle primer sequences required genotyping each genetically modified mouse line.
  • Electrophoresis gels were run at 100 V for 60 - 80 minutes until adequate band separation had occurred and imaged using a UV light source.
  • MOP results we expect to see a WT band visible at 700 bp and a KO band visible at 400 bp.
  • DOP reaction we expect to observe a band at 1000 bp and a KO band at 600 bp.
  • the BAR2 reaction produced a WT band at 188 bp and a KO band at 400 bp.
  • For all of the reactions one band was present to represent either WT or KO and heterozygote animals were identified by the presence of both bands.
  • the hot water tail withdrawal assay was used to assess morphine analgesia.
  • An electronic thermostatic circulating water bath (Thermostatic circulator bath Optima general purpose digital +5°C to 100°C, 12L stainless steel tank available from Fisher Scientific) was used to maintain a temperature within ⁇ 0.1 °C.
  • a baseline tail withdrawal assay was performed using 48°C water with a maximum exposure time of 15 seconds. Any suitable tail withdrawal assay technique can be applied to establish a baseline.
  • the tail assay method used in these experiments is a standard procedure, as reported by Lam H, Maga M, Pradhan A, Evans CJ, Maidment NT, Hales TG, Walwyn W. 201 1 .
  • mice were treated with cumulative doses of morphine sulphate (Sigma-Aldrich) at 0.1 , 0.3, 1 , 3, 10 and 30 mg/Kg, with the morphine sulphate being passed through a 0.2 pm syringe filter prior to use.
  • morphine sulphate Sigma-Aldrich
  • references to morphine in relation to the experimental results herein mean morphine sulphate which may be diluted as appropriate according to the requirements of the particular experiment.
  • the NaCI solution containing morphine sulphate suitable for a 30mg/Kg was diluted down to provide a solution for the lower dose levels in order that the mice were dosed with the same volume in each instance
  • Morphine was diluted in 0.9% NaCI to provide a solution suitable for injection and the mice were dosed via subcutaneous injections (SC). Thirty minutes after each morphine dose the tail withdrawal assay was performed. Results were calculated as a percentage of maximal possible effect (MPE) where the % MPE 100*(drug latency - basal latency) / (15 s - basal latency). Once a mouse had reached the 15 second maximum it received no further doses of drug. Morphine tolerance
  • mice were treated with 10 mg/Kg morphine sulphate sc once daily for 10 days. The injections were performed at the same time each day and all experiments took place during the light phase. On each experimental day a baseline tail withdrawal assay was performed using the circulating hot water bath (bath settings 48°C, 15 s maximum exposure time). Mice then received a 1 mg/Kg injection of morphine sulphate administered subcutaneously and a repeat tail withdrawal assay was performed 30 minutes later. Following this they received an injection of 10 mg/Kg morphine sulphate administered subcutaneously, with a repeat tail withdrawal assay again performed thirty minutes after the morphine dose.
  • One chamber had a wallcovering consisting of black and white horizontal stripes and the other black and white vertical stripes.
  • the floor material was the same in each of the chambers; it consisted of 1 cm square wire grid flooring material.
  • the grid direction of this material differs depending on its orientation. We utilised this property to provide a difference in the floor between each chamber.
  • the direction of the grid matched the wall stripe direction in each chamber.
  • Each test arena measured 28 x 28 cm and was 19 cm high.
  • Two test arenas, each consisting of a two compartment apparatus, are contained within an operant box. The test apparatus was matched to mice of specific genders and only mice of the same gender were placed in the same operant box set-up.
  • mice placed in the same operant box for testing were cage mates, but this could not always be ensured for the male mice.
  • These boxes are soundproofed and allowed the light levels to be controlled at approximately 70 lumens, the temperature of the room was maintained between 21 and 23°C.
  • the matched vehicle injection contained the same constituents but without the active drug, PP2, commercially available from Tocris Bioscience, Bristol, United Kingdom, was also dissolved in DMSO at its solubility limit of 25 mM and diluted in a 0.9% saline solution to give a final concentration of 1 mg/ml (16% DMSO and 16% Kolliphor EL).
  • the inactive compound, PP3, 1-phenyl-1 H-pyrazolo[3,4-c ]pyrimidin-4-amine Both PP2 and PP3 are commercially available from Tocris Bioscience, Bristol, United Kingdom, has a higher solubility limit and was made up in DMSO at 100 mM and diluted in a 0.9% saline solution to give a final concentration of 1 mg/ml (5% DMSO and 5% Kolliphor® EL). These were all administered via the intraperitoneal (IP) route.
  • IP intraperitoneal
  • MOP-/- mice The Applicant has confirmed in MOP-/- mice that morphine (10 mg/Kg) does not cause analgesia in the absence of MOP receptors. Morphine also does not inhibit VACC's in DRG neurones from MOP-/- mice. In MOP+/- mice the Applicant observed a reduced analgesic potency of morphine with a rightward shift in the dose response curve, whilst there did not appear to be a change in efficacy of the drug in opioid naive mice. This reduction in potency is significant, while the slope of the morphine dose response relationship remained unchanged.
  • Table 2 provides a summary of the results obtained and in particular morphine ED 50 (mg/Kg) and slope values for morphine analgesia in WT, MOP+/-, DOP+/-, DOP-/-, BAR2-/- and BAR2-/-//DOP-/- mice. * P ⁇ 0.05 on Student's t test compared to WT.
  • MOP+/- mice exhibited a very rapid onset of tolerance following daily treatments with 10 mg/Kg morphine. They have significantly less analgesia at day 4 of the study protocol compared to day 1 . This contrasts with WT mice in which there was no significant difference in morphine analgesia until day 9 of the protocol. MOP+/- mice also developed a significantly greater degree of tolerance to the analgesic effects of morphine by day 10 than that observed for WT mice. These results support the proposal that MOP receptor number is important in the development of tolerance to morphine's analgesic effects. It is known that MOP+/- mice have 50% fewer MOP receptors than WT mice due to the deletion of one copy of the oprml gene.
  • the slope of the morphine analgesia dose response relationship is also significantly reduced in the DOP-/- mice compared to the WT mice. Without wishing to be bound to any particular theory we propose that this could be explained by a difference in the way that morphine interacts with MOP/DOP receptor oligomers compared to homomeric MOP receptors on nociceptive neurones. It is possible that binding of morphine to MOP receptors is influenced by occupancy of adjacent DOP receptors leading to cooperativity.
  • BAR2-/- mice did develop tolerance to morphine over the course of our 10 day protocol, but this was significantly reduced when compared to the WT mice.
  • BAR2-/- mice show basal analgesia, their baseline tail withdrawal times, in the absence of exogenously administered drug, are significantly prolonged when compared to the WT mice.
  • DOP-/- mice baseline tail withdrawal times are not significantly prolonged when compared to those of the WT mice. From our results it appears that the removal of BAR2 allows constitutive signalling of MOP receptors to occur resulting in basal analgesia. There does not appear to be an involvement of DOP receptors in this process.
  • MOP receptors are also required for the reinforcing effects of morphine.
  • WT mice did not display a preference for the morphine paired chamber following conditioning with morphine 3 mg/Kg at any of the time points. There were no significant differences in the time spent in each chamber across the test period. This is also the case for the MOP-/- mice conditioned with morphine (10 mg/Kg). These results support our proposal that MOP receptors are important in this process, and in particular that MOP receptors alone are responsible for this effect and that receptor number may have a role in this process.
  • Our results for the DOP-/- mice exhibited no significant alteration in locomotor activation at 10 mg/Kg morphine compared to the WT mice but interestingly did demonstrate increased sensitivity to its locomotor activating effects. They exhibited significant locomotor activation at the 3 mg/Kg dose which the WT mice do not. These results imply that the DOP receptor may be involved in limiting the sensitivity of the locomotor system to activation by morphine.
  • BAR2-/-//DOP-/- mice also exhibited significantly reduced locomotor activation following administration of morphine 10 mg/Kg compared to WT mice. This is not significantly different to that observed in the BAR2-/- mice. These mice do not exhibit a significant preference for morphine following conditioning with morphine 3 mg/Kg, although there is a trend towards significance. They did demonstrate a preference for the morphine paired chamber following conditioning with morphine 10 mg/Kg. They exhibited no extinction of preference during the test period at either dose and their preference scores for morphine were not significantly different from those of the BAR2-/- mice.
  • c-Src inhibitors The effects of c-Src on opioid receptor signalling As detailed hereinbefore PP2 and dasatanib as c-Src inhibitors. We used these compounds to investigate the effects of c-Src inhibition on locomotor activation by morphine and conditioned place preference to morphine. We also used PP2 to investigate the effects of c-Src inhbition and MEK inhibition on sIPSC frequency in the VTA.
  • Dasatinib when administered prior to morphine, prevents the development of significant morphine tolerance in both WT and MOP+/- mice. It does not significantly alter basal tail withdrawal latency, this is interesting because BAR2-/- mice exhibit prolonged basal tail withdrawal latencies and this suggests that another signalling pathway may be responsible for this aspect of behaviour.
  • PP2 also inhibits the development of morphine analgesic tolerance; this inhibition is not significantly different from that observed following the administration of dasatinib.
  • Dasatinib also reversed the analgesic tolerance that had already developed to morphine in the MOP+/- mice. The observed reversal and return to almost full analgesia suggests that these effects can be rapidly reversed and implicated c-Src in these processes.
  • Dasatinib had no psychomotor effects when administered alone. Mice administered either vehicle or dasatinib injections prior to morphine exhibited a reduced locomotor response. This may be due to an effect of the components of the vehicle injection (DMSO / Kolliphor EL®) on locomotor activity or a behavioural effect due to a further period of restraint and injection. We have demonstrated that inhibition of c-Src does not produce reinforcement or aversion in the absence of morphine. Furthermore, when mice were treated with either vehicle or dasatinib prior to morphine during the conditioning phase of CPP there was no significant differences in the preference for the morphine paired chamber on test day. Dasatinib does not appear to affect morphine reinforcement.
  • BAR2-/- mice which exhibited an increased sensitivity to morphine reinforcement compared to the WT mice.
  • BAR2-/- mice exhibited a significant preference for morphine following conditioning with morphine at both 3 and 10 mg/Kg. This was not the case for WT mice or for WT mice administered dasatinib prior to morphine. In those mice reinforcement was only exhibited following conditioning with morphine at a higher dose (10mg/Kg), but not the lower dose (3mg/Kg). This suggests that c-Src is not implicated in this process of sensitisation to the reinforcing effects of morphine.
  • the c-Src inhibitor, PP2 reduced the ability of morphine to inhibit sIPSC frequency in VTA neurones.
  • the diminished sIPSC inhibition by morphine in the presence of PP2 was similar to that observed in recordings from BAR2-/- neurones.
  • PP3 an inactive chemical analogue of PP2
  • BAR2 and c-Src are important components of the signalling pathway within the VTA in response to morphine.
  • Systemic administration of the MEK inhibitor SL 327 has been demonstrated to inhibit locomotion in a dose dependent manner that involves D1 dopamine receptors in WT mice.
  • SW620 cells were incubated with PP2, dasatinib and PP3 (10 ⁇ ) and DMSO control for 24 hours prior to collection.
  • Primary antibodies were used against actin (loading control) total c- Src and phosphorylated c-Src (pc-Src). Both PP2 and dasatinib reduced pc-Src with DMSO and PP3 had no effect.
  • Vertical lines represent ⁇ SEM. *, p ⁇ 0.05, **, p ⁇ 0.01 , ***, p ⁇ 0.001 .
  • Additional data (as discussed in relation to the Figures herein) demonstrates that c-Src inhibition inhibits morphine tolerance in MOP+/- mice.
  • Dasatinib does not cause basal analgesia in MOP+/- mice.
  • PP2 does not cause basal analgesia in MOP+/- mice.
  • PP2 (5mg/Kg) administered IP 30 minutes prior to subcutaneous morphine (10 mg/Kg) administration prevented the morphine tolerance observed in the PP3 (5 mg/Kg IP) treated mice, two way repeated measures ANOVA, time p ⁇ 0.0001 , drug difference p ⁇ 0.0001 , post hoc Bonferrroni results are shown on the graph.
  • Dasatinib reversed the development of tolerance in MOP+/- mice.
  • mice 16 mice (5 male and 3 female in each group) were treated with morphine (10 mg/Kg) daily for three days, on days 4 and 5 one group received dasatinib (5 mg/Kg IP) 30 minutes prior to subcutaneous morphine administration and the other group received a vehicle injection at the equivalent time.
  • the mice that received dasatinib exhibited a significantly greater morphine analgesia compared to the vehicle treated mice; two way repeated measures ANOVA revealed a significant difference between the two groups on days 4 and 5.
  • the post hoc Bonferroni test results are shown on graph. Vertical lines represent ⁇ SEM. *, p ⁇ 0.05, **, p ⁇ 0.01 , ***, p ⁇ 0.001 .

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Abstract

La présente invention concerne l'utilisation d'inhibiteurs de c-Src pour l'atténuation d'effets secondaires associés à l'analgésie opioïde et en particulier pour l'atténuation de la tolérance aux opioïdes en médecine humaine et vétérinaire.
PCT/GB2017/051361 2016-05-16 2017-05-16 Traitement de la tolérance aux opioïdes WO2017199014A1 (fr)

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