WO1998016230A1 - Procedes permettant d'inhiber de maniere reversible la myelopoiese dans des tissus mammaliens - Google Patents

Procedes permettant d'inhiber de maniere reversible la myelopoiese dans des tissus mammaliens Download PDF

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WO1998016230A1
WO1998016230A1 PCT/US1997/018951 US9718951W WO9816230A1 WO 1998016230 A1 WO1998016230 A1 WO 1998016230A1 US 9718951 W US9718951 W US 9718951W WO 9816230 A1 WO9816230 A1 WO 9816230A1
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alkyl
optionally substituted
aryl
crior20
compound
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PCT/US1997/018951
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English (en)
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Daniel Horowitz
Andrew G. King
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Smithkline Beecham Corporation
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Priority to AU51475/98A priority Critical patent/AU5147598A/en
Publication of WO1998016230A1 publication Critical patent/WO1998016230A1/fr

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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

Definitions

  • the present invention relates to the field of myelopoiesis in mammalian tissue, and more particularly, to the use of certain imidazoles containing compounds for reversibly inhibiting myelopoiesis in humans and animals.
  • Certain chemotherapeutic and radiation treatments for cancer and other serious illness have the undesired side effect of killing all dividing and differentiating cells. This effect is most severe on the differentiating cells of the hematopoietic system, which are essential for repopulating the bone marrow and permitting the redevelopment of a functioning immune system following such treatment.
  • bone marrow toxicity or myelosuppression is the limiting factor in the use of such treatments.
  • the acute effects of myelosuppression are the loss of neutrophils (neutropenia) and the loss of platelets (thrombocytopenia) in the blood. These conditions in part increase patient susceptibility to infections and hemorrhagic complications.
  • a variety of regulatory messengers and modifiers such as colony stimulating factors, interferons, and different types of compounds are responsible for the regulation of myelopoiesis, which is the development and growth of early progenitor cells (stem cells) of the hematopoietic system.
  • the present invention provides for a method for reversibly inhibiting myelopoiesis in mammals, mammalian tissue or other samples in vivo and ex vivo comprising administering to said animal, tissue, or sample an effective amount of a myeloprotectant compound of formula (I) below.
  • the amount of the compound is effective to reversibly inhibit the formation of myelopoietic colonies during the time the animal, tissue, or sample is exposed to myelosuppressive therapy.
  • Figure 2 demonstrates a dose response effect of (IP administered) Compounds 2 and 3 on WBC counts 9 days post 5-FU in the mouse; *P ⁇ 0.05.
  • Figure 3 demonstrates a dose response effect of (IP administered) Compounds 2 and 3 on PMN counts 9 days post 5-FU in the mouse; *P ⁇ 0.05.
  • Figure 4. demonstrates a dose response effect of (IP administered) Compounds 2 and
  • Figure 5 demonstrates a dose response effect of (IP administered) Compounds 2 and 3 on lymphocyte counts 9 days post 5-FU in the mouse; *P ⁇ 0.05.
  • Figure 6 demonstrates a dose response effect of (IP administered) Compounds 2 and
  • Figure 9 demonstrates a dose response effect of (PO administered) Compounds 2 and 3 on PMN cell counts 9 days post 5-FU in the mouse.
  • Figure 10 demonstrates a dose response effect of (PO administered) Compounds 2 and 3 on bone marrow cell counts 9 days post 5-FU in the mouse. Marrow counts obtained from pools of 3 mice/group.
  • Figure 11 demonstrates the effect of Compounds 2 and 3 and G-CSF on the kinetics of neutrophil recovery post 175 mg/kg 5-FU in mice. G-CSF (50ug kg) administered daily IP, animals were bleed 1.5 hours post G-CSF injection on the indicated days.
  • Figure 12 demonstrates the effect of Compounds 2 and 3, and G-CSF on the kinetics of monocyte recovery post 175 mg/kg 5-FU in mice.
  • G-CSF 50ug/kg administered daily IP, animals were bleed 1.5 hours post G-CSF injection on the indicated days.
  • Figure 13 demonstrates the effect of Compounds 2 and 3, and G-CSF on the kinetics of platelet recovery post 175 mg/kg 5-FU in mice.
  • G-CSF (50ug/kg) administered daily IP animals were bleed 1.5 hours post G-CSF injection on the indicated days.
  • Figure 14 demonstrates the effect of Compounds 2 and 3, and G-CSF on the kinetics of lymphocyte recovery post 175 mg kg 5-FU in mice.
  • G-CSF (50ug/kg) administered daily IP animals were bleed 1.5 hours post G-CSF injection on the indicated days.
  • Figure 15 demonstrates the effect of Compounds 2 and 3, and G-CSF on the kinetics of total white blood cell recovery post 175 mg/kg 5-FU in mice.
  • mice 5-FU in mice.
  • G-CSF 50ug/kg administered daily IP, animals were bleed 1.5 hours post G-CSF injection on the indicated days.
  • Figure 18 demonstrates the effect of Compound 3, G-CSF, and the combination of
  • Figure 19 demonstrates the effect of Compound 3, G-CSF, and the combination of
  • Figure 20 demonstrates the effect of Compound 3, G-CSF, and the combination of Compound 3 + G-CSF on the kinetics of total white blood cell recovery post
  • This invention provides for a method for reversibly inhibiting myelopoiesis in mammals, mammalian tissue, including bone marrow and other samples, in vivo and ex vivo.
  • This method entails administering to the mammal, tissue or sample an amount of a compound of formula (I) effective to reversibly inhibit the formation of myelopoietic colonies during the time the mammal, tissue or bone marrow is exposed to myelosuppressive therapy.
  • the myelopoietic colonies inhibited are CFU-C colony forming cells.
  • the myelopoietic CFU-C colony forming cells as used herein include, but are not limited to, CFU-G, CFU-M, CFU-GM, CFU-GEMM, CFU-Meg, and HPP.
  • CFU colony forming unit
  • G granulocyte
  • M macrophage or megakaryocyte
  • E erythroid colony types
  • HPP high proliferative potential colony.
  • a preferred colony forming cell is CFU-GM.
  • This method may be accomplished in vivo.
  • a mammalian subject preferably a human patient, undergoing chemotherapy or radiation may be administered the compound so that the reversible inhibition of myelopoiesis occurs in vivo.
  • Treatment with a myeloprotectant compound of this invention may be necessary throughout the time period in which the insulting agent is present within the body. Inhibition of normal hematopoietic stem cells during this period could protect normal cells while damaging only target cells such as cancer cells.
  • the compounds of this invention have demonstrated reversible inhibition, as demonstrated in the Example Section below, a more rapid rebound of hematopoietic stem cell proliferation is expected when administration of the myeloprotectant compound is ceased (concurrently with, or shortly after administration of the chemotherapeutic or radiation is ceased).
  • the end result of treatment with the compounds of this invention as myeloprotective agents is to prevent neutropenia and or accelerate neutrophil / leukocyte recovery, and prevent stem cell loss due to myelosuppression.
  • the advantage of such administration to the patient includes fewer infections due to leukopenia and the ability to use more aggressive chemotherapy regimens to kill cancer cells (dose intensification / increase dose frequency).
  • cytokine inhibitors as described in USSN 08/091,491, published as WO95/02575; WO96/21452; US Patent No.: ; USSN 08/473,396; US Patent No. 5,658,903 ; USSN 08/764,003; USSN 08/473,398; WO96/21654; WO93/14081; US Patent 5,656,644;USSN 08/095,234; US 5,656,644; WO95/03297; USSN 08/481,671; PCT/US97/00619; PCT/US97/00614; PCT/US97/00500; PCT/US 97/00529; USSN 60/013,357; USSN 60/013,358; USSN 60/013,359; WO93/14082; WO95/13067 ;WO95/31451 WO95/13067;WO95/31451 WO95/31451 WO 97/05877; WO 97/
  • a preferred group of compounds for use herein are those compounds of the formula (I):
  • Rl is 4-pyridyl, pyrimidinyl, 4-pyridazinyl, l,2,4-triazin-5-yl, quinolyl, isoquinolinyl, quinazolin-4-yl, 1-imidazolyl or 1-benzimidazolyl ring, which ring is optionally substituted independently one to three times with Y.
  • NHR a optionally substituted -4 alkyl, halogen, hydroxyl, optionally substituted C ⁇ -4 alkoxy, optionally substituted Ci-4 alkylthio, Ci-4 alkylsulfmyl, CH2OR12, amino, mono and di- -6 alkyl substituted amino, or N(R ⁇ o)C(O)R D ;
  • Y is O-R a ;
  • R4 is phenyl, naphth-1-yl or naphth-2-yl, or a heteroaryl, which is optionally substituted by one or two substituents, each of which is independently selected, and which, for a 4-phenyl, 4-naphth-l-yl, 5-naphth-2-yl or 6-naphth-2-yl substituent, is halogen, cyano, nitro, C(Z)NR7Ri7, C(Z)ORi6, (CRioR20)vCORi2, SR5, SOR5, OR12, halo-substituted-Ci-4 alkyl, Ci-4 alkyl, ZC(Z)Ri2, NRioC(Z)Ri6, or (CRioR2 ⁇ )vNRl ⁇ R20 and which, for other positions of substitution, is halogen, cyano, C(Z)NRi3Ri4, C(Z)OR3, (CRioR20)m"COR3, S(
  • R2 is hydrogen, (CRioR20)n' OR9, heterocyclyl, heterocyclylCi-io alkyl,
  • R a is Ci-6alkyl, aryl. arylC i-6alkyl. heterocyclic, heterocyclylC i -6 alkyl, heteroaryl, or heteroarylC i-6alkyl, wherein each of these moieties may be optionally substituted;
  • Rb is hydrogen, C ⁇ . ⁇ alkyl, C3-7 cycloalkyl, aryl, arylCi- 4 alkyl, heteroaryl, heteroarylC i-4alkyl, heterocyclyl, or heterocyclylC 1 -4 alkyl;
  • R3 is heterocyclyl, heterocyclylC i-io alkyl or R ⁇ ;
  • R5 is hydrogen, Ci-4 alkyl, C2-4 alkenyl, C2-4 alkynyl or NR7R17, excluding the moieties -SR5 being -SNR7R17 and -SOR5 being -SOH;
  • R6 is hydrogen, a pharmaceutically acceptable cation, Ci
  • Ci-10 alkanoyl is each independently selected from hydrogen or C 1-4 alkyl or R7 and
  • Rl7 together with the nitrogen to which they are attached form a heterocyclic ring of 5 to 7 members which ring optionally contains an additional heteroatom selected from oxygen, sulfur or NR15;
  • R8 is Ci-io alkyl, halo-substituted Ci-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C3-7 cycloalkyl, C5-7 cycloalkenyl, aryl, arylCi-io alkyl, heteroaryl, heteroarylC l- 10 alkyl, (CRioR20)nORl l, (CR ⁇ oR2 ⁇ )nS(O) m Ri8, (CRioR20)nNHS(O)2Rl8,
  • R9 is hydrogen, C(Z)Ri 1 or optionally substituted Ci-io alkyl, S(O)2Rl8, optionally substituted aryl or optionally substituted aryl-Ci-4 alkyl;
  • RlO and R20 is each independently selected from hydrogen or C 1-4 alkyl;
  • Rl 1 is hydrogen, Ci-10 alkyl, C3-7 cycloalkyl, heterocyclyl, heterocyclyl
  • Ci-4 alkyl optionally substituted aryl or optionally substituted aryl-Ci-4 alkyl, or together with the nitrogen to which they are attached form a heterocyclic ring of 5 to 7 members which ring optionally contains an additional heteroatom selected from oxygen, sulfur or NR9;
  • Ri5 is Rio or C(Z)-Ci-4 alkyl;
  • Rl6 is Ci-4 alkyl, halo-substituted-Cl-4 alkyl, or C3.7 cycloalkyl
  • Rl8 is C i-io alkyl. C3-7 cycloalkyl, heterocyclyl, aryl, arylalkyl, heterocyclyl, heterocyclyl-Ci-ioalkyl, heteroaryl or heteroarylalkyl;
  • Rl9 is hydrogen, cyano, C 1 - 4 alkyl, C3. ⁇ cycloalkyl or aryl; or a pharmaceutically acceptable salt thereof.
  • Ri is a substituted 4-pyridyl or 4-pyrimindyl. More suitably R, is substituted by alkoxy, alkylthio, amino, methylamino, NHRa, or Y.
  • a preferred ring placement of the Ri substituent on the 4-pyridyl derivative is the 2-position, such as 2-methoxy -4-pyridyl.
  • a preferred ring placement on the 4-pyrimidinyl ring is also at the 2-position, such as in 2-methoxy-pyrimidinyl.
  • the substituent is Y, and Ra is aryl, it is preferably phenyl or naphthyl.
  • R a is aryl alkyl, it is preferably benzyl or napthylmethyl.
  • the heterocyclic portion is preferably pyrrolindinyl, piperidine, morpholino, tetrahydropyran, tetrahydrothiopyranyl, tetrahydrothipyran-sulfinyl, tetrahydrothio-pyransulfonyl, pyrrolindinyl, indole, or piperonyl.
  • the heterocyclic rings herein may contain unsaturation, such as in a tryptamine ring.
  • the aryl, heterocyclic and heteroaryl rings may be optionally substituted one or more times independently with halogen; Ci-4 alkyl, such as methyl, ethyl, propyl, isopropyl, or t-butyl; halosubstituted alkyl, such as CF3; hydroxy; hydroxy substituted Ci-4 alkyl; Cj-4 alkoxy, such as methoxy or ethoxy; S(O) m alkyl and S(O)m aryl (wherein m is 0, 1, or 2); C(O)OR ⁇ 1, such as C(O)Ci-4 alkyl or C(O)OH moieties; C(O)R ⁇ 1; OC(O)R c ; O-(CH2)s-O-, such as in a ketal or dioxyalkylene bridge; amino; mono- and di-Ci-6 alkylsubstituted amino; N(R ⁇ o)C(O)R D ; C(O)NR
  • R c is optionally substituted -6 alkyl, C3.7 cycloalkyl, aryl, arylCi- 4 alkyl, heteroaryl, heteroarylC i-4alkyl, heterocyclyl, or heterocyclylC 1.4 alkyl moieties.
  • the R a groups include Cj_4 alkyl, benzyl, halosubstituted benzyl, napthylmethyl, phenyl, halosubstituted phenyl, aminocarbonylphenyl, alkylphenyl, cyanophenyl.
  • alkylthiophenyl hydroxyphenyl, alkoxyphenyl, mo holinopropyl, piperonyl, piperidin-4-yl, alkyl substituted piperidine, such as 1 -methyl piperidine, or 2,2,6,6-tetramethylpiperidin-4-yl.
  • R a is aryl, arylalkyl, halosubstituted arylalkyl, halosubstituted aryl, heterocyclic alkyl, hydroxy alkyl, alkyl- 1-piperidine-carboxylate, heterocyclic, alkyl substituted heterocyclic, halosubstituted heterocyclic, or aryl substituted heterocyclic.
  • R is benzyl, halosubstituted benzyl, napthylmethyl, phenyl, halosubstituted phenyl, mo ⁇ holinopropyl, 2-hydroxy ethyl, ethyl- 1-piperidinecarboxy late, piperonyl, piperidin-4-yl, alkyl substituted piperidine, chlorotryptamine, and tetrathiohydropyranyl.
  • the alkyl chain is substituted by halogen, such as fluorine, chlorine, bromine or iodine; hydroxy, such as hydroxyethoxy; Ci-io alkoxy, such as a methoxymethoxy, S(O)m alkyl, wherein m is 0, 1 or 2; amino, mono & di- substituted amino, such as in the NR7R17 group, i.e.
  • halogen such as fluorine, chlorine, bromine or iodine
  • hydroxy such as hydroxyethoxy
  • Ci-io alkoxy such as a methoxymethoxy, S(O)m alkyl, wherein m is 0, 1 or 2
  • amino, mono & di- substituted amino such as in the NR7R17 group, i.e.
  • R2 is selected from hydrogen, Ci-io alkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylC l- 10 alkyl, (CRioR20)nNS(O)2Rl8, (CRioR20)nS(O) m Ri8, arylCi-io alkyl,
  • R2 is mo ⁇ holino propyl, piperidine, N-methylpiperidine, N-benzylpiperidine, 2,2,6,6-tetramethylpiperidine, 4-aminopiperidine, 4-amino-2,2,6,6-tetramethyl piperidine, 4-hydroxycyclohexyl, 4- methyl-4-hydroxy cyclohexyl, 4-pyrrolinindyl-cyclohexyl, 4-methyl-4- aminocyclohexyl, 4-methyl-4-acetamidocyclohexyl, 4-keto cyclohexyl, 4-oxiranyl, or 4-hydroxy-4-( 1 -propynyl)cyclohexyl.
  • R2 is hydrogen, optionally substituted heterocyclyl ring, optionally substituted heterocyclylC l- 10 alkyl, optionally substituted -io alkyl, optionally substituted C3-7cycloalkyl, optionally substituted C3-7cycloalkyl Ci-io alkyl, (CR ⁇ oR2 ⁇ )nC(Z)OR ⁇ 1 group, (CRioR20)nNRi3Rl4,
  • R2 is an optionally substituted heterocyclyl ring, and optionally substituted heterocyclylC l- 10 alkyl, optionally substituted aryl,
  • the ring is preferably a mo ⁇ holino, pyrrolidinyl, or a piperidinyl group.
  • the substituents may be directly attached to the free nitrogen, such as in the piperidinyl group or pyrrole ring, or on the ring itself.
  • the ring is a piperidine or pyrrole, more preferably piperidine.
  • the heterocyclyl ring may be optionally substituted one to four times independently by halogen; Ci-4 alkyl; aryl, such as phenyl; aryl alkyl, such as benzyl - wherein the aryl or aryl alkyl moieties themselves may be optionally substituted (as in the definition section below);
  • C(O)ORi such as the C(O)Ci-4 alkyl or C(O)OH moieties; C(O)H; C(O)C ⁇ _4 alkyl, hydroxy substituted Ci-4 alkyl, -4 alkoxy, S(O) m Ci-4 alkyl (wherein m is 0, 1, or 2), NRl ⁇ R20 (wherein Rio and R20 are independently hydrogen or Cl- 4alkyl).
  • the ring is a piperidine, the ring is attached to the imidazole at the 4-position, and the substituents are directly on the available nitrogen, i.e.
  • R2 is an optionally substituted heterocyclyl Ci-io alkyl group
  • the ring is preferably a mo ⁇ holino, pyrrolidinyl, or a piperidinyl group.
  • this alkyl moiety is from 1 to 4, more preferably 3 or 4, and most preferably 3, such as in a propyl group.
  • Preferred heterocyclic alkyl groups include but are not limited to, mo ⁇ holino ethyl, mo ⁇ holino propyl, pyrrolidinyl propyl, and piperidinyl propyl moieties.
  • the heterocyclic ring herein is also optionally substituted in a similar manner to that indicated above for the direct attachment of the heterocyclyl.
  • R2 is an optionally substituted C3-7cycloalkyl, or an optionally substituted C3-7cycloalkyl Ci-io alkyl
  • the cycloalkyl group is preferably a C4 or C6 ring, most preferably a C6 ring, which ring is optionally substituted.
  • the cycloalkyl ring may be optionally substituted one to three times independently by halogen, such as fluorine, chlorine, bromine or iodine; hydroxy; Ci-10 alkoxy, such as methoxy or ethoxy; S(O)m alkyl, wherein m is 0, 1, or 2, such as methyl thio, methylsulfinyl or methyl sulfonyl; S(O)m aryl; cyano, nitro, amino, mono & di- substituted amino, such as in the NR7R17 group, wherein R7 and R17 are as defined in Formula (I), or where the R7R17 may cyclize together with the nitrogen to which they are attached to form a 5 to 7 membered ring which optionally includes an additional heteroatom selected from oxygen, sulfur or NR15 (and R 15 is as defined for Formula (I)); N(R ⁇ o)C(O)X ⁇ (and Xi is Ci-4 alkyl, aryl or arylC
  • R e is a 1,3-dioxyalkylene group of the formula -O-(CH2)s-0-, wherein s is 1 to 3, preferably s is 2 yielding a 1 ,3-dioxyethylene moiety, or ketal functionality.
  • R6' is NRi9'R20' a ⁇ Y ⁇ l-6> halosubstituted alkyl 1-6; hydroxy substituted alkyl 1-6; alkenyl 2-6; aryl or heteroaryl optionally substituted by halogen, alkyl 1-6, halosubstituted alkyl 1 -6, hydroxyl, or alkoxy i- ⁇ .
  • R20 ' is H, alkyl 1.5, aryl, benzyl, heteroaryl, alkyl substituted by halogen or hydroxyl, or phenyl substituted by a member selected from the group consisting of halo, cyano, alkyl ⁇ _ 12, alkoxy 1. , halosubstituted alkyl 1-6, alkylthio, alkylsulphonyl, or alkylsulfinyl; or R19' and R20' may together with the nitrogen to which they are attached form a ring having 5 to 7 members, which members may be optionally replaced by a heteroatom selected from oxygen, sulfur or nitrogen. The ring may be saturated or contain more than one unsaturated bond.
  • R6' is NRi9'R20' and R19' and R20' are preferably hydrogen. When the R2 cycloalkyl moiety is substituted by NR7R17 group, or NR7R17
  • the substituent is preferably an amino, amino alkyl, or an optionally substituted pyrrolidinyl moiety.
  • Preferred compounds of Formula (I) include: 4-(4-Fluorophenyl)-5-(2-amino-4-pyridiminyl)- 1 -( 1 -methyl-4- piperidinyl)imidazole;
  • halogen such as fluorine, chlorine, bromine or iodine
  • hydroxy hydroxy substituted Ci-ioalkyl
  • Ci-io alkoxy such as methoxy or ethoxy
  • S(O)m alkyl wherein m is 0, 1 or 2, such as methyl thio, methylsulfinyl or methyl sulfonyl
  • amino, mono & di-substituted amino such as in the NR7R17 group; or where the R7R17 may together with the nitrogen to which they are attached cyclize to form a 5 to 7 membered ring which optionally includes an additional heteroatom selected from O/N/S; - io alkyl, cycloalkyl, or cycloalkyl alkyl group, such as methyl, ethyl, propyl, isopropyl, t-butyl, etc.
  • halosubstituted Cl-10 alkyl such CF3
  • an optionally substituted aryl such as phenyl, or an optionally substituted arylalkyl, such as benzyl or phenethyl, wherein these aryl moieties may also be substituted one to two times by halogen; hydroxy; hydroxy substituted alkyl; Cl-10 alkoxy; S(O) m alkyl; amino, mono & di-substituted amino, such as in the NR7R17 group; alkyl, or CF3.
  • compounds of formula (I) may be administered parenterally, that is by intravenous, intramuscular, subcutaneous intranasal, intrarectal, intravaginal or intraperitoneal administration.
  • the subcutaneous and intramuscular forms of the parenteral form administration are generally preferred.
  • Appropriate dosage forms for such administration may be prepared by conventional techniques.
  • An oral administration is the most preferred route of treatment.
  • the effective amounts or dosages may be adjusted based on the amount of tissue being treated.
  • an effective in vitro concentration is in the range of about 10 ⁇ 9 M to 10 " 5 M.
  • One of skill in the art may readily determine other appropriate dosages, depending on the mode of administration, and the level of aggressiveness of therapy required in the specific circumstance.
  • One screening and identification method useful in the present invention may employ the step of screening test samples which detectably bind to a receptor for in vitro or in vivo inhibition of CFU-C colony formation in either a conventional 7 day CFU-GM assay (CFU-C Assay), well known to those skilled in the art, or in the novel, micro-screening assay as described herein.
  • CFU-C Assay 7 day CFU-GM assay
  • the micro-inhibition assay is a modification of the conventional assay and provides for an efficient and rapid screening and identification of inhibitors. The presence or amount of inhibition of CFU-C or CFU-GM colony formation can then be measured in order to identify those test samples which act as agonists.
  • PCLC pre-CFU-liquid culture
  • the assay methods may be further modified by preparing two or more additional test samples from the original test sample or samples that are determined to inhibit CFU-C colony formation activity. These additional test samples contain a lesser number of test compounds than the original test sample from which they were prepared. The steps of the assays may then be repeated as many times as desired or until the test compound or compounds which bind to the receptor preparation have been identified.
  • the Micro-Reversibility assay of this invention is performed as follows. 100,000 non-adherent murine bone marrow cells/ml were incubated for 24 hours with compound ( 5 uM, 0.5 uM, 50 nM, 5 nM) and a mixture of CSFs (mixture of IL-1, IL-3, SCF, M-CSF) in McCoys 5a liquid culture medium with 15% FBS. After 24 hours the cells were washed and resuspended in media / agar / and M-CSF and assayed according to the Micro-Inhibition assay of Example 1, above.
  • the present invention designed studies to prove whether the compounds of Formula (I) were reversible inhibitors of CFU-GM and hence myeloprotective in vivo in a high dose 5-FU model as described herein.
  • Stock solutions are diluted in PBS prior to injection.
  • the test compound concentration is adjusted so that a 0.2 ml injection delivers the appropriate dose of compound.
  • mice Female BDF1 mice were injected IP, (or the dosage may be given orally) with 10 mg/kg 1 hour prior to and 3 hours after 175 mg/kg 5-FU. Mice were sacrificed on days 3, 4, and 6 post 5- FU injection. Bone marrow cellularity and CFU-GM analysis was performed on each day. Data shown in Figure 1 demonstrate that 5-FU severely depleted the marrow compartment of CFU-GM evident on days 3 and 4. By day 6 recovery has initiated, however CFU-GM content is less than 5% of normal.
  • Example 4 A Myeloprotectant Compound Effects Stem Cells Post 5-Fluorouracil Myelosuppression
  • Compound 3 also significantly increased PMN in a dose related manner.
  • both compounds increased platelet counts in a dose responsive manner. Platelet numbers in the 5-FU model, decrease by 60% than rebound to values higher than normal on days 10 - 15. In this experiment, the 5-FU and normal platelets counts are equivalent as the 5-FU counts are beginning this rebound phase of platelet production. Both compounds resulted in higher PLT counts at day 9 indicating that PLT recovery was quicker in the CSAIDTM compound treated mice.
  • Compounds 2 and 3 significantly increased lymphocyte, monocyte, and RBC counts in a dose responsive manner. These data taken together indicate that multiple hematopoietic cell lineage's are protected by administration of compounds of Formula (I) in this murine 5-FU model.
  • mice were injected IP with compound 1 hour prior to and 3 hours after the cell cycle specific drug, 5- fluorouracil (5-FU 175mg/kg), which occurred at 0 hour.
  • 5- fluorouracil 5- fluorouracil
  • G-CSF (50 ug/kg administered daily 48 hours post 5-FU) is included as a positive control for enhanced PMN recovery in these studies. It should be noted that G-CSF is administered 1.5 hours before each bleed point. G-CSF administration under these conditions reflects PMN recovery plus additional PMN mobilization from vascular and possibly tissue sites.
  • Compounds of Formula (I) have shown efficacy as myeloprotectants in a murine high dose 5-FU model noted above.
  • Compound 3 40 mg/kg was administered IP to mice 2 hours before and 4 hours after a single injection of 175 mg/kg 5-FU G-CSF was administered daily starting 48 hours post 5-FU injection for 10 days.

Abstract

Nouvelle utilisation de composés de formule (I) en tant que composés myéloprotecteurs dont l'activité biologique inhibe de manière réversible la myélopoïèse dans des tissus mammaliens et des échantillons biologiques in vitro, ex vivo ou in vivo.
PCT/US1997/018951 1996-10-17 1997-10-17 Procedes permettant d'inhiber de maniere reversible la myelopoiese dans des tissus mammaliens WO1998016230A1 (fr)

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EP0961618A1 (fr) * 1996-12-11 1999-12-08 Smithkline Beecham Corporation Nouveaux composes
EP1082320A1 (fr) * 1998-05-26 2001-03-14 Smithkline Beecham Corporation Nouveaux composes d'imidazole substitue
US6362193B1 (en) 1997-10-08 2002-03-26 Smithkline Beecham Corporation Cycloalkenyl substituted compounds
US6489325B1 (en) 1998-07-01 2002-12-03 Smithkline Beecham Corporation Substituted imidazole compounds
US6503930B1 (en) 1997-05-22 2003-01-07 G.D. Searle & Company Pyrazole derivatives as p38 kinase inhibitors
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US6548503B1 (en) 1998-11-04 2003-04-15 Smithkline Beecham Corporation Pyridin-4-yl or pyrimidin-4-yl substituted pyrazines
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US6617324B1 (en) 1997-05-22 2003-09-09 G. D. Searle & Company Substituted pyrazoles as p38 kinase inhibitors
US6852740B2 (en) 1997-05-22 2005-02-08 G. D. Searle & Co. Pyrazole derivatives as p38 kinase inhibitors
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