EP1730100A1 - Polyamine analogs that activate antizyme frameshifting - Google Patents
Polyamine analogs that activate antizyme frameshiftingInfo
- Publication number
- EP1730100A1 EP1730100A1 EP04821940A EP04821940A EP1730100A1 EP 1730100 A1 EP1730100 A1 EP 1730100A1 EP 04821940 A EP04821940 A EP 04821940A EP 04821940 A EP04821940 A EP 04821940A EP 1730100 A1 EP1730100 A1 EP 1730100A1
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- methyl
- ethyl
- hydrogen
- aliphatic
- substituted
- Prior art date
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C211/00—Compounds containing amino groups bound to a carbon skeleton
- C07C211/01—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to acyclic carbon atoms
- C07C211/26—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to acyclic carbon atoms of an unsaturated carbon skeleton containing at least one six-membered aromatic ring
- C07C211/27—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to acyclic carbon atoms of an unsaturated carbon skeleton containing at least one six-membered aromatic ring having amino groups linked to the six-membered aromatic ring by saturated carbon chains
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
- C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
- C07D211/08—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms
- C07D211/18—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms
- C07D211/26—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms with hydrocarbon radicals, substituted by nitrogen atoms
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
- C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
- C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
- C07D213/36—Radicals substituted by singly-bound nitrogen atoms
- C07D213/38—Radicals substituted by singly-bound nitrogen atoms having only hydrogen or hydrocarbon radicals attached to the substituent nitrogen atom
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
- C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
- C07D233/64—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms, e.g. histidine
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
- C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
- C07D277/04—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
Definitions
- the present invention relates to novel polyamines, their synthesis and use in pharmacological, cosmetic or agricultural applications.
- the instant invention provides polyamines that induce antizyme production which in turn down regulates both the production of polyamines by ornithine decarboxylase (ODC) and the transport of polyamines by its corresponding polyamine transporter. These compounds will preferably enter the cell independent of the polyamine transporter.
- ODC ornithine decarboxylase
- these compounds will preferably enter the cell independent of the polyamine transporter.
- drugs these compounds are used to treat any disease associated with cellular proliferation including but not limited to cancer. As such, they will be useful as drags to treat diseases where components of the immune system undergo undesired proliferation. These compounds will also be effective for the treatment of unwanted proliferation of hair on skin.
- the present invention also identifies key structural elements expected to comprise the antizyme inducing motif(s) of small molecules related to polyamines.
- Polyamines are essential for cell proliferation through their involvement in DNA replication, cell cycle regulation, and protein synthesis. Depletion of intracellular polyamine levels inhibits cell growth. Antizyme regulates polyamine levels both by inhibiting polyamine biosynthesis and uptake/import. The importance of their function is highlighted by the fact that specific biosynthesis, degradation, uptake and excretion pathways tightly control cellular polyamine levels (Heby, O. Differentiation 19:1-20 (1981); Seiler, N. et. al, Int. J. Biochem. 22:211-218 (1990); Seiler, N. et. al, J. P. Int. J. Biochem. Cell Biol. 28:843-861 (1996)).
- ODC Ornithine decarboxylase
- Putrescine is then converted to both spermidine and spermine by the sequential transfer of an aminopropyl group from decarboxylated -S- adenosylmethionine.
- Increasing concentrations of intracellular polyamine levels induce the production of antizyme which negatively regulates ODC by binding to it and targeting it for destruction.
- Antizyme has also been shown to inhibit polyamine uptake (Mitchell, J. L. et. al, Biochem. J. 299:19-22 (1994); Suzuki, T. et. al, Proc. Natl Acad. Sci. USA 91: 8930-8934 (1994); Sakata, K. et. al, Biochem. Biophys. Res.
- antizyme may increase polyamine excretion (Sakata, K. et. al, Biochem J. 347:297-303 (2000)). Therefore, antizyme can very effectively limit the accumulation of cellular polyamines. Antizyme has been found in vertebrates, fungi, nematodes, insects and eukaryotes (Ivanov, I. et. al, Nucleic Acids Res. 28:3185-3196 (2000)). Three antizyme isoforms, AZ1, AZ2 and AZ3, have now been identified among vertebrates. Both AZ1 and AZ2 have wide tissue distribution but AZ2 mRNA is less abundantly expressed.
- AZ3 is expressed only in the testis germ cells (Ivanov, I. et. al, Proc. Natl. Acad. Sci. USA 97: 4808-4813 (2000); Tosaka, Y. et. al, Genes to Cells 5:265-276 (2000)) where expression begins early in spermiogenesis and finishes in the late spermatid phase.
- Antizyme production is controlled by a unique regulatory mechanism known as translational frameshifting (Matsufuji, S. et. al, Cell 80: 51-60 (1995)).
- the antizyme gene consists of two overlapping open reading frames (ORFs). The bulk of the coding sequence is encompassed in the second (ORF2) but it does not contain an initiation codon.
- ORF1 is short but contains two AUG initiation codons. Either one of the initiation codons can be used to initiate translation but normally little full length mRNA is made unless a +1 frameshift occurs just before the ORFl UGA stop codon enabling translation to continue. Only minute quantities of antizyme are generally present in mammalian tissues. Polyamines and agmatine have been found to greatly enhance the efficiency of frameshifting (Hayashi, S. et. al, Trends Biochem. Sci. 21:27-30 (1996); Satriano, J. et. al, J. Biol Chem. 273:15313-15316 (1998)).
- Antizyme binding disrupts the homodimer interface leading to the formation of two antizyme-ODC heterodimers that are now enzymatically inactive (Kameji, T. et. al, Biochim. Biophys. Acta 717:111-117 (1982); Kern, A.D. et. al, Struct. Fold. Des. 7:567-581 (1999)).
- Antizyme directs the ODC monomer to the proteosome where it is degraded without ubiquitination (Murakami, Y. et. al, Nature 360:597- 599 (1992); Tokunaga, F. et. al, J. Biol Chem. 269:17382-17385 (1994)).
- Antizyme is then released and free to interact with and destroy additional ODC monomers in a catalytic fashion.
- the AZ2 isoform has not been shown to catalytically induce the degradation of ODC, although AZ2 has been shown to inhibit both ODC and polyamine uptake equipotently (Zhu, C. et. al, J. Biol. Chem. 21 A: 26425-26430 (1999).
- AZ3 is the most recently discovered antizyme and has also been shown to inhibit ODC (Ivanov, I. et. al, Proc. Natl Acad. Sci. USA 97:4808-4813 (2000); Tosaka, Y. et. al, Genes to Cells 5:265-276 (2000)).
- Antizyme is regulated by antizyme inhibitor, which has a higher affinity towards antizyme than ODC (Fujita, K. et. al, J. Biol Chem. 274:26424-26430 (1982); Kitani, T. et. al, Biochim. Biophys. Acta 991:44-49 (1989); Murakami, Y. et. al, Biochem. J. 259:839-845 (1989)). Thus it may rescue ODC from degradation by displacing it from antizyme.
- Antizyme inhibitor like ODC, forms a homodimer and has a high degree of sequence homology with ODC. However, it does not form heterodimers with ODC (Murakami, Y. et. al.
- Frameshifting efficiency is determined by comparing the ratio of firefly luciferase to renilla luciferase activity in cells transfected in parallel using a control vector containing a constitutive +1 frameshift (AZ-IF) that measures the in- frame translation efficiency and a vector containing the inducible 0 to +1 frameshift (AZ1) construct.
- AZ-IF constitutive +1 frameshift
- AZ1 constitutive +1 frameshift
- AZ1 constitutive +1 frameshift
- AZ1 constitutive +1 frameshift
- this assay system gives a quantitative measure of the efficiency of the polyamines, polyamine analogs and other compounds to induce frameshifting in a cell-based bioassay.
- Cells must be pretreated with ⁇ -difluoromethylomithine (DFMO), an irreversible inhibitor of ODC, prior to screening to decrease the basal antizyme frameshifting levels and increase the sensitivity to polyamine or compound-mediated stimulation of antizyme frameshifting.
- DFMO ⁇ -difluoromethylomithine
- ODC ⁇ -difluoromethylomithine
- oligoamines such as octamines, decamines and dodecamines were found to induce antizyme to varying degrees (Mitchell, J. L. A. et. al, Biochem.
- Polyamines may arrest prostate cell growth in the Gl phase by inducing antizyme.
- the prostate is the only vertebrate organ that synthesizes polyamines for export. As such, this tissue is exposed to higher concentrations of the polyamines.
- Spermine has been found to be a naturally occurring inhibitor of prostatic carcinoma cell growth in vitro and in vivo (Smith, R. C. et. al, Nature Med. 1:1040-1045 (1995)). Subsequently, it was found that spermine could induce Gl arrest in poorly metastatic prostatic carcinomas but not in highly malignant cells (Koike, C et. al, Cancer Res. 59:6109-6112, (1999)). Furthermore, antizyme could be induced only in the poorly metastatic prostatic carcinomas.
- Antizyme was later found to affect the cell- cycle of prostatic carcinoma cells with the discovery that it could interact with Gl cyclin Dl and its associated cyclin-dependent kinase, cdk4 (Coffino, P. Nat. Rev. Mol. Cell. Biol. 2:188-194 (2001)).
- the degradation of cdk4 and cyclin Dl were dependent on antizyme and independent of ubiquitin using in vitro purified proteasomes.
- the steady-state levels of the cyclin and kinase decreased when the polyamine levels were experimentally raised in the cultured cells. It has been proposed that prostatic cells that lose the ability to activate antizyme may eventually become malignant (Koike, C et. al, Cancer Res.
- ODC In mice, ODC is expressed in ectodermal cells at sites where hair follicles develop during embryonic development (Nancarrow, M.J., et. al, Mech. Dev. 84: 161-164 (1999); Schweizer, J. In: Molecular Biology of the Skin: The Keratinocyte, Darmon M, et. al., Eds., Academic Press, New York, 1993, pp 33-78).
- ODC protein expression is down regulated when the hair follicle enters catagen and is not detected in telogen. ODC protein expression does not resume until new follicle formation commences.
- ODC vibrissae
- SSAT is a key enzyme in the catabolism of polyamines that is rate-limiting for the conversion of spermine to spermidine and spermidine to putrescine.
- Both transgenic animal models showed permanent hair loss in which the normal hair follicles were transformed into dermal cysts that progressively increased in size as the animals aged (Pietila, M. et. al, J. Biol. Chem. 272:18746-18751 (1997); Suppola, S. et. al, Biochemistry 7338:311-316 (1999); Soler, A.P. et. al, J. Invest. Dermatol.
- DFMO treatment of mice challenged with tumor allografts resulted in modified cytotoxic T-lymphocyte and antibody responses (Ehrke, J. M. et. al, Cancer Res. 46:2798-2803 (1986)).
- Reports by Singh et al. indicate that DFMO treatment may also ameliorate acute lethal graft versus host (ALGVH) disease in mice (Singh, A. B. et. al, Clin.Immunol. Immunopathol 65:242-246 (1992)).
- Murine ALGVH represents a model of human GVH that contributes to the morbidity and mortality of bone marrow transplantation in humans and is characterized by anemia and the loss of T cell function and numbers.
- epithelial cells of the mouth or intestine become damaged and depleted, thinning and ulceration can result (mucositis) leading to pain and potential infection. Oral mucositis is also the result of damaged stem cells. Oral tissues are particularly painful if damaged. Under normal conditions, the lining of the intestine is continuously being renewed through the proliferation of epithelial stem cells and their progeny in the crypts of villi (Booth, D, et. al, JNatl Cancer Inst Monogr 29:16-20 (2001)). When damage occurs (e.g., radiation or cytotoxic insult), a burst of proliferation/regeneration occurs in undamaged stem cells.
- damage e.g., radiation or cytotoxic insult
- Ideal polyamine analogs should not substitute for the normal physiological functions of polyamines such as having the ability to rescue cells from DFMO-induced growth inhibition in vitro. It is also desirable that these compounds not be readily metabolized to regenerate polyamines. Identifying compounds that induce frameshifting and ultimately increase full length antizyme protein levels will be useful for depleting intracellular polyamine levels. These compounds should be an effective therapy for any disease associated with cellular proliferation including but not limited to cancer. As such, they are useful as drugs in a number of diseases where components of the immune system undergo undesired proliferation.
- Non-limiting examples include asthma, inflammation, autoimmune diseases, psoriasis, restenosis, rheumatoid arthritis, scleroderma, systemic and cutaneous lupus erythematosus, Type I insulin dependent diabetes, tissue transplantation, osteoporosis, hyperparathyroidism, treatment of peptic ulcer, glaucoma, Alzheimer's disease, Crohn's disease and other inflammatory bowel diseases.
- Other disease states associated with the proliferation of fungal, bacterial, viral and parasitic agents such as African sleeping sickness are also included. These compounds will also be effective for the treatment of unwanted proliferation of hair on skin.
- Antizyme inducers will be useful in the treatment of diseases involving the cell cycle by pausing the cell cycle progression during radiation or chemotherapy treatment. The appropriate cells will accumulate in G 0 or Gi, protecting them from radiation or chemotherapy induced hair loss (alopecia) and mucositis.
- Figure 1 is a tabular representation of a large number of polyamine analogs A-S (25 ⁇ M) that were tested for their ability to induce antizyme frameshifting using the dual luciferase reporter assay.
- Figure 2 shows the frameshifting induced by 25 ⁇ M of various compounds in HEK-
- Figure 3 shows the dose-dependent induction of frameshifting in HEK-293 cells with various compounds.
- Figure 4 shows the growth inhibition of HEK-293 cells with compound A.
- Figure 5 gives a comparison of the ability of antizyme frameshifters (25 ⁇ M) to rescue cells from 2.5 mM DFMO-induced growth inhibition compared to 25 ⁇ M spermidine (SPD) in a 6-day assay in HEK-293 cells.
- Figure 6 is a graph showing the effect of a 6-day incubation of compound A on HEK-293 cellular polyamine levels and cell growth.
- Figure 7 illustrates the effect of extracellular compound A on the intracellular concentration of compound A in HEK-293 cells as determined by HPLC.
- Figure 9 shows the reaction scheme for the synthesis of compound B.
- Figure 10 shows the reaction scheme for synthesis of intermediate R groups for Figure 11.
- Figure 11 shows the reaction scheme for the synthesis of compounds C-R.
- n can be 0 to 8 and the aminomethyl functionality can be ortho, meta or para substituted
- R is hydrogen, -CH 3 , -CH 2 CH 3 , 2-aminoethyl, 3-aminopropyl, 4-aminobutyl, 5- aminopentyl, 6-aminohexyl, 7-aminoheptyl, 8-aminooctyl, N-methyl-2-aminoethyl, N-methyl-3- aminopropyl, N-methyl-4-aminobutyl, N-methyl-5-aminopentanyl, N-methyl-6-aminohexyl, N- methyl-7-aminoheptyl, N-methyl-8-aminooctyl, N-ethyl-2-aminoethyl, N-ethyl-3-aminopropyl, N-ethyl-4-aminobutyl, N-ethyl-5-aminopentyl, N
- n can be 0 to 8
- R and Rj are described as above
- R 2 can be independently selected from hydrogen, -CH 3 or -CH 2 CH 3 and R 3 and may be the same or different and are independently selected from hydrogen, or flourine.
- An additional preferred embodiment of compounds that can be used according to this disclosure are described in formula III:
- n and n can be 0 to 7 independently, but m cannot equal n when Ri equals R 2 and R 3 equals R 4 , o can be 2 to 4, R can be independently selected from H, -CH 3 or -CH 2 CH 3 , Ri and R 2 can be independently selected from hydrogen, -CH 3 or -CH 2 CH 3 and R 3 and R 4 may be . the same or different and are independently selected from hydrogen or fluorine.
- Another aspect of the present invention are compounds of formula IV:
- R is hydrogen, -CH 3 , or -CH 2 CH 3 , m and n can be 0 to 7 independently and o can be 2 to 4, R 2 can be independently selected from hydrogen, -CH 3 or -CH 2 CH 3 and R 3 and R 4 may be the same or different and are independently selected from hydrogen or fluorine.
- compounds of the present invention are represented by formula V
- R is hydrogen, -CH 3 , or -CH 2 CH 3
- m can be 0 to 7
- n can be 0 to 8 and o can be 2 to 4
- R 2 can be independently selected from hydrogen, -CH 3 or -CH 2 CH 3 and R 3 and R may be the same or different and are independently selected from hydrogen or fluorine.
- the present disclosure also relates to novel compounds of formulae I, II, III, IV and V . above with the further proviso that the novel compounds are non-symmetrical substituted xylene derivatives. In other words, differs from
- R3 R4 RHN. . differs from RHN. 1 ⁇ / m
- Preferred novel compounds according to the present disclosure are those compounds wherein only one side of the xylene ring contains a group other than -CH 2 NH 2 with the most preferred compounds being B, T and U as shown in Figure 1.
- the polyamine analogs decrease cellular polyamine levels and can be used to
- the invention additionally encompasses the stabilization of polyamine analogs by modifying them to resist enzymatic degradation. Such modifications include substitution of primary amine groups with alkyl groups, the addition of alkyl groups to the terminal amino groups and the addition of fluorine atoms ⁇ to the terminal amino groups.
- polyamine analogs of the invention enter cells by pathways other than those of active polyamine transport regulated by antizyme.
- an additional embodiment of the invention are analogs that are not imported into cells primarily by the polyamine transporters. Frameshifting activity is only one of the requirements for a good antizyme inducer. According to the present invention, it is preferable that the compounds enter
- the present invention also relates to pharmaceutical compositions comprising an effective amount of at least one of the above disclosed compounds.
- a further aspect of the present invention relates to treating a condition associated with cellular proliferation by administering at least one of the compounds described above.
- the present disclosure also relates to treating one or more conditions associated with cellular proliferation comprising administration of at least one of B, T or U shown in Figure 1.
- conditions include, but are not limited to condition is selected from the group consisting of cancer, mucositis, asthma, inflammation, autoimmune disease, psoriasis, restentosis, rheumatoid arthritis, scleroderma, systemic and cutaneous lupus erythematosus, Type I insulin dependent diabetes, tissue transplantation, osteoporosis, hyperparathyroidism, treatment of peptic ulcer, glaucoma, Alzheimer's disease, and inflammatory bowel diseases.
- the administration can be systemic, for example, and can be oral.
- the administration can be via a time-release vehicle, if desired.
- the compounds R and S can be formulated as a cosmetic.
- Another aspect of the present invention relates to inhibiting hair growth comprising topical administration of at least one of B, T or U shown in Figure 1 to a subject in need of hair growth inhibition.
- the present invention also relates to of inhibiting hair loss (alopecia) comprising topical administration of at least one of B, T or U shown in Figure to a subject undergoing radiation or chemotherapy.
- aryl refers to monocyclic or bicyclic aromatic hydrocarbon groups having 6 to 12 carbon atoms in the ring portion, such as phenyl, naphthyl, biphenyl and diphenyl and diphenyl groups, each of which may be substituted.
- alkyl refers to straight or branched chain unsubstituted hydrocarbon groups of 1 to 20 carbon atoms, preferably 1 to 8 carbon atoms.
- lower alkyl refers to unsubstituted alkyl groups of 1 to 4 carbon atoms.
- suitable alkyl groups include methyl, ethyl and propyl.
- branched alkyl groups include isopropyl and t-butyl.
- halogen or “halo” refers to fluorine, chlorine, bromine and iodine.
- the alkoxy groups typically contains about 1 - 8 carbon atoms and more typically about 1 - 4 carbon atoms. Examples of suitable alkoxy groups are methoxy, ethoxy and propoxy. Examples of some suitable alkaryl groups include phenyl C 1-3 alkyl such as benzyl.
- substitution groups are NO 2 , alkyl, CF 3 , alkoxy and halo.
- suitable cycloalkyl groups typically contain 3-8 carbon atoms and include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
- fused bicyclic unsatured ring groups are 2-quinolinyl, 3-quinolinyl, 5- quinolinyl, 6-quinolinyl, 7-quinolinyl, 1 -isoquinolinyl, 3-isoquinolinyl, 6-isoquinolinyl, 7- isoquinolinyl, 3-cinnolyl, 6-cinnolyl, 7-cinnolyl, 2-quinazolinyl, 4-quinazolinyl, 6-quinazolinyl, 7-quinazolinyl, 2-quinoxalinyl, 5-quinoxalinyl, 6-quinoxalinyl, 1-phthalaonyl, 6-phthalazinyl, 1- 5-naphthyridin-2-yl, l,5-naphthyridin-3-yl, l,6-naphthyridin-3-yl, l,6-naphthyridin-7
- Substitutions for each of the fused ring groups include the above noted group of substituents described herein.
- Examples of mono- and multi-ring groups include aryl and bicyclic fused aryl-cycloalkyl groups.
- the aryl groups include an aromatic substituent which can be a single ring of multiple rings (up to three rings) which are fused together or linked covalently.
- the rings may each contain from zero to four heteroatoms selected from N, O and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quarternized.
- Non- limiting examples of aryl groups include phenyl, 1 -naphthyl, 2-naphthyl, biphenyl, 1-pyrrolyl, 2- pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5- benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2- quinoxalin
- substitutions for each of the above noted aryl systems include the above noted group of substitutents described herein.
- the "bicyclic fused aryl-cycloalkyl” groups are those groups in which an aryl ring (or rings) is fused to a cycloalkyl group (including cycloheteroalkyl groups. The group can be attached to the remainder of the molecule through either an available valence on the aryl portion of the group, or an available valence on the cycloalkyl portion of the group. Examples of such benzotetrahydropyranyl and 1,2,3,4-tetrahydronaphthyl. Substitutents for each of the above noted groups include the group of substituents described herein.
- the compounds of the present invention can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic agents or in a combination of therapeutic agents. They can be administered alone, but generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.
- the present invention includes the free base or acid forms, as well as salts thereof, of the polyamines and derivatives described by the above formulas.
- the invention also includes the optical isomers of the above described analogs and derivatives.
- mixtures of enantiomers and/or diastereoisomers, resulting from a single preparative step, combination, or interconversion are encompassed.
- Prodrug forms of the compounds bearing various nitrogen functions may include the following types of derivatives where each R group individually may be hydrogen, substituted or unsubstituted alkyl, aryl, alkenyl, alkynyl, heterocycle, alkylaryl, aralkyl, aralkenyl, aralkynl, cycloalkyl or cycloalkenyl groups as defined beginning on Page 7.
- Another prodrug derived from a carboxylic acid form of the invention may be a quaternary salt type R of structure described by Bodor et al.. J. Med. Chem. 1980, 23, 469. It is of course understood that the compounds of the present invention relate to all optical isomers and stereo-isomers at the various possible atoms of the molecule.
- the compounds used in the methods of this invention form pharmaceutically acceptable acid and base addition salts with a wide variety of organic and inorganic acids and bases and includes the physiologically acceptable salts which are often used in pharmaceutical chemistry. Such salts are also part of this invention.
- Typical inorganic acids used to form such salts include hydrochloric, hydrobromic, hydroiodic, nitric, sulfuric, phosphoric, hypophosphoric and the like.
- Salts derived from organic acids such as aliphatic mono and dicarboxylic acids, phenyl substituted alkonic acids, hydroxyalkanoic and hydroxyalkandioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, may also be used.
- Such pharmaceutically acceptable salts thus include acetate, phenylacetate, trifluoroacetate, acrylate, ascorbate, benzoate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate, o-acetoxybenzoate, naphthalene-2-benzoate, bromide, isobutyrate, phenylbutyrate, ⁇ -hydroxybutyrate, butyne- 1 ,4-dioate, hexyne- 1 ,4-dioate, cabrate, caprylate, chloride, cinnamate, citrate, formate, fumarate, glycollate, heptanoate, hippurate, lactate, malate, maleate, hydroxymaleate, malonate, mandelate, ' mesylate, nicotinate, isonicotinate, nitrate, oxalate, phthalate, teraphthalate,
- Bases commonly used for formation of salts include ammonium hydroxide and alkali and alkaline earth metal hydroxides, carbonates, as well as aliphatic and primary, secondary and tertiary amines, aliphatic diamines.
- Bases especially useful in the preparation of addition salts include sodium hydroxide potassium hydroxide, ammonium hydroxide, potassium carbonate, methylamine, diethylamine, and ethylene diamine.
- the invention also provides prodrug forms of the above described compounds and derivatives, wherein the prodrug is metabolized in vivo to produce a compound or derivative as set forth above. Indeed, some of the above described compounds or derivatives may be a prodrug for another compound or derivative.
- the prodrug is a substrate for either spermidine synthase or spermine synthase which are enzymes that transfer an aminopropyl group to putrescine and spermidine, respectively.
- the compounds may be utilized alone or in combination with other agents, particularly other inhibitors of polyamine synthesis or transport, but including other inhibitors of cell proliferation.
- the polyamines compound of the invention may decrease polyamine levels by inducing antizyme (AZ), which in turn down regulates both the production of polyamines by ornithine decarboxylase (ODC) and the transport of polyamines by its corresponding transporter. Polyamine levels may also decrease because antizyme may induce increases in polyamine excretion.
- the invention further defines structural elements/motifs within these compounds that appear key to their induction of antizyme as determined by assays. Because polyamines are absolutely essential for DNA replication and are essential to cellular homeostasis, there is an interest in preventing cell proliferation by lowering intracellular polyamine levels. Sufficiently low polyamine levels can lead to cell death. Thus any agent able to lower polyamine levels, particularly by inhibiting both polyamine biosynthesis and uptake/import, offers the opportunity to target a variety of disease or undesirable conditions related to cell proliferation, including cancer.
- the compounds of the invention are not necessarily metabolized like naturally occurring polyamines. As such, the compounds of the invention may have the advantage of not being readily metabolized to regenerate polyamines.
- compositions containing the above described compounds and derivatives are provided.
- the compositions are formulated to be suitable for pharmaceutical or agricultural use by the inclusion of appropriate carriers or excipients.
- Figure 1 is a tabular representation of a large number of polyamines A-S (25 ⁇ M) that were tested for their ability to induce antizyme frameshifting using the dual luciferase reporter assay.
- the percent relative frameshifting value (% RF) gives a comparison of the ability of a compound to induce frameshifting compared to 25 ⁇ M spermidine.
- the % RF was calculated as follows. The background percent frameshifting activity determined from the 2.5 mM DFMO negative control was subtracted from the percent frameshifting activity for all compounds including the spermidine control. The background corrected frameshifting activity of each compound was then divided by the background corrected frameshifting activity induced by 25 ⁇ M spermidine and multiplied by 100.
- the compounds were also evaluated for their ability to rescue cells from DFMO-induced growth inhibition by determining their rescue coefficient.
- the rescue coefficient represents the ratio of the cell growth, as measured by O.D., in the presence of the test compound with 2.5 mM DFMO to the growth in the presence of 2.5 mM DFMO alone.
- the frameshift-rescue factor (FRF) is a useful factor to compare the effectiveness of the various antizyme frameshifters by taking into account their potency for inducing frameshifting and ability to rescue cells from DFMO-induced growth inhibition.
- the FRF was calculated by multipling the %RF value by the inverse of the rescue coefficient.
- Figure 2 shows the frameshifting induced by 25 ⁇ M of various compounds in HEK-293 cells.
- FIG. 3 shows the dose-dependent induction of frameshifting in HEK-293 cells with various compounds. Values represent percent frameshifting following transient transfection of an AZl-IF control and the inducible 0 to +1 AZl plasmid constract into HEK-293 cells grown in the presence of 2.5 mM DFMO. The compounds were added to the cells after transfection and incubated overnight before assaying the next day. Each value represents triplicate measurements.
- Figure 4 shows the growth inhibition of HEK-293 cells with compound A.
- HEK-293 cells were incubated with 1 mM aminoguanidine and various concentrations of compound A with or without 1 ⁇ M spermidine during a 6-day growth assay.
- Cell number was determined by MTS/PMS assay from triplicate wells.
- Figure 5 gives a comparison of the ability of antizyme frameshifters (25 ⁇ M) to rescue cells from 2.5 mM DFMO-induced growth inhibition compared to 25 ⁇ M spermidine (SPD) in a 6-day assay in HEK-293 cells.
- the rescue coefficient represents the ratio of cell growth, as measured by O.D., of the test compound with 2.5 mM DFMO versus 2.5 mM DFMO alone.
- Figure 6 is a graph showing the effect of a 6-day incubation of compound A on HEK-293 cellular polyamine levels and cell growth. All flasks received 1 mM aminoguanidine. Cells were washed, counted, lysed in perchloric acid, dansylated and polyamine levels determined by HPLC. Each value represents the average of triplicate values with the error bars representing standard deviation.
- Figure 7 illustrates the effect of extracellular compound A on the intracellular concentration of compound A in HEK-293 cells as determined by HPLC. Cells were prepared as in Figure 6. Peak area was normalized by dividing the peak area of the compound divided by the peak area of the internal standard, diaminoheptane. Values represent at least triplicate measurements with the error bars representing standard deviation.
- Figure 8 shows the reaction scheme for the synthesis of compound A.
- Figure 9 shows the reaction scheme for the synthesis of compound B.
- N-(3-aminopropyI)-xylyIenediamin (Compound A) - To the clear homogeneous solution of 1.97 g (10.4 mmole) of N-(2-cyanoethyl)-xylylenediamine in 50 mL of dry THF is added 30 mL (30 mmole) of a 1M solution of lithium aluminum hydride (LiAlH ) in THF dropwise at 25°C under an atmosphere of argon. Bubbles form and a white precipitate forms immediately. A pink color is noted.
- LiAlH lithium aluminum hydride
- Resin 2.3 All of the resin from the step are suspended in 50 mL of CH 2 C1 2 in a 100 mL solid-phase peptide synthesis vessel and treated with 9.31 g (42 mmole, 3 equiv) of 2- nitrobenzenesulfonyl chloride and 5.85 mL (4.25 g, 42 mmole, 3 equiv) of triethylamine at 25°C.
- the resin is agitated via the introduction of a slow stream of argon through the bottom frit of the vessel for 4 h when the resin is filtered and washed with CH 2 C1 2 , j PrOH, DMF, THF then CH 2 C1 2 (3 x 75 mL each).
- Resin 2.7 To all of the resin 2.6 obtained above suspended in 30 mL of dry THF is added as solids 11.02 g (42 mmole, 3 equiv) of triphenylphosphine and 8.62 g (42 mmole, 3 equiv) of N-(3-hydroxypropyl)phthalimide. The resulting suspension is agitated with a slow stream of argon through the bottom of the vessel while 8.49 g (42 mmole, 3 equiv) of diisopropylazodicarboxyate is introduced in a dropwise fashion.
- Resin precursor to Compounds C through R (Resin 4.2) -
- a 100 mL solid-phase peptide synthesis vessel containing 10 g (Rapp Polymer 14 mmole, 1.4 mmole/g) of polystyrene- based trityl chloride resin in 30 mL CH 2 C1 2 is added a solution of 10.51 g (140 mmole, 10 equiv) of 3-aminopropanol in 30 mL of CH 2 C1 2 dropwise at 25°C over 30 min.
- the resin is agitated via the introduction of a slow stream of argon through the bottom frit of the vessel.
- the reaction is allowed to proceed for 5 h.
- the resin is filtered and washed with CH 2 C1 2 , j PrOH, DMF, THF then CH 2 C1 2 (3 x 75 mL each).
- the dried resin is treated with 10 mL of diethylamine in 40 mL of CH 2 C1 2 for 2 h to completely cap the resin.
- the same washing procedure as above followed by a thorough overnight vacuum drying process take place to give the product resin.
- This resin gives a negative Kaiser amine test reaction.
- Resin 4.2 To a 0.25 gram portion of Resin 4.2 obtained above is added 10 mL each of EtOH and hydrazine hydrate. The resin is heated at 60°C for 18 as for Resin 2.5 above. After 18 h at this temperature the vessel is allowed to cool and the resin is filtered and washed with H 2 O, EtOH, CH 2 C1 2 , 'PrOH, DMF, THF then CH 2 C1 2 (3 x 75 mL each). The resin is dried under vacuum overnight to give a product that is positive by the Kaiser amine test reaction.
- the resulting resin is treated with 0.435 g (3.15 mmole, 9 equiv) of K 2 CO 3 and 0.32 mL (3.15 mmole, 9 equiv) of thiophenol in 10 mL of dry DMF at 25°C for 3 h.
- the resin is filtered and washed with CH 2 C1 2 , 'PrOH, DMF, THF then CH 2 C1 2 (3 x 15 mL each) to gives a product that gives a positive Kaiser amine test reaction.
- This resin is then suspended in 10 mL of TFA/CH 2 Cl 2 /'Pr 3 SiH 20:78:2 and agitated for 30 min. on a platform shaker.
- EXAMPLE 4 Cell Culture and Reagents All cell lines are obtained from ATCC (Manassas, VA) and cultured in the recommended media, serum, and CO 2 concentration. Medias are obtained from Mediatech, Inc. (Herndon, WA) and serums from Gibco BRL (Gaithersburg, MD). 50 U/mL penicillin, 50 mg/mL streptomycin and 2 mM L-glutamine (all from BioWhittaker, Walkersville, MD) are included in all cultures.
- HEK-293 cells are plated in white sided, clear-bottomed 96-well assay plates at 15,000 cells per well in 100 ⁇ L of medium (DMEM supplemented with 10% fetal bovine serum (Gibco), 1% penicillin, streptomycin and L-glutamine) containing 2.5 mM DFMO.
- the cells are incubated for 2-days at 37°C in an atmosphere of 5% CO 2 .
- the medium is removed and the cells are then transfected with lipofectamine reagent (LifeTechnologies) using serum free DMEM. All cells are transfected overnight with 100 ng of the appropriate plasmid DNA and 0.3 ⁇ L lipofectamine in 50 ⁇ L of serum free DMEM containing 2.5 mM DFMO.
- the next day, 40 ⁇ L of medium is added per well containing 3.1 mM DFMO, 2.5 mM aminoguanidine and 25% FBS.
- Compounds are diluted in either water or medium to 0.25 mM and 10 ⁇ L is added per well such that the final concentration is 25 ⁇ M.
- the positive control contains 25 ⁇ M spermidine and for the negative control no compound is added.
- the cells are incubated overnight at 37°C in an atmosphere of 5% CO 2 , washed once with IX PBS, lysed with 50 ⁇ L of passive lysis buffer (Promega) and assayed for renilla and firefly luciferase activity using the Dual-Luciferase Reporter Assay System (Promega).
- the percent frameshifting activity is determined by dividing the ratio of the firefly luciferase to renilla luciferase activity in cells transfected with the inducible 0 to +1 AZl construct by the ratio of the firefly luciferase to renilla luciferase activity in cells transfected with the control vector AZ-IF and multiplied by 100.
- the percent relative frameshifting value (% RF) gives a comparison of the ability of a compound to induce frameshifting compared to 25 ⁇ M spermidine.
- the % RF is calculated as follows.
- the background percent frameshifting activity determined from the 2.5 mM DFMO ' negative control is subtracted from the percent frameshifting activity for all compounds including the spermidine control.
- the background corrected frameshifting activity of each compound is then divided by the background corrected frameshifting activity induced by 25 ⁇ M spermidine and multiplied by 100. From the above screenings, a number of compounds are found to induce frameshifting
- Fig. 1 and Fig. 2 Some of these compounds induce frameshifting substantially better than spermidine at the same concentration (25 ⁇ M).
- the polyamines spermine, spermidine, putrescine and compound A are also titrated using the dual luciferase reporter assay (Fig. 3).
- EC 50 represents the concentration of the compound that resulted in 50% of the maximum percent frameshifting as measured by the plateau values in Figure 3.
- the titration indicates that EC 50 values for induction of frameshifting of all three polyamines are close to 1 ⁇ M with a rank order potency of spermidine (0.56 ⁇ M) > spermine (0.68 ⁇ M) > putrescine (0.95 ⁇ M).
- Compound A has a less potent EC 50 for frameshifting of about 120 ⁇ M.
- the maximum levels of frameshifting also vary between putrescine, spermidine, spermine and compound A. It has generally been reported that the rank order potency for frameshift induction by the polyamines beginning with the most potent is spermine, followed by spermidine then putrescine. However in this study (Fig.
- both putrescine and spermidine can be converted intracellularly into spermidine and spermine via the transfer of an aminopropyl group by spermidine synthase or spermine synthase, respectively. It is conceivable the frameshifting activities observed for putrescine and spermidine may actually reflect a combination of activities for the polyamines synthesized during the course of the assay.
- EXAMPLE 6 Growth Inhibition Assay Cells are plated in 96-well plates such that they would be in log growth for the duration of the assay. The day after plating, the polyamine analogs are added to the cells, and growth, if any, permitted to continue for six days in the presence of 1 mM AG and 1 ⁇ M SPD when appropriate. At the end of six days, cell growth is measured by MTS/PMS dye assay (Cell Titer 96 Aqueous Non-Radioactive Cell Proliferation Assay; Promega, Madison, WI). IC 50 refers to the concentration of the polyamine analog that results in 50% of maximum cell growth inhibition. The cell growth of HEK-293 cells is inhibited by compound A (Fig. 4).
- EXAMPLE 7 Polyamine Analogs and Rescue from DFMO induced growth inhibition Compounds found to induce antizyme frameshifting are evaluated for the undesired ability to rescue cells from DFMO-induced growth inhibition (Fig. 1 and Fig. 5).
- HEK-293 cells are plated in 96-well assay plates at 1,000 cells per well in 100 ⁇ L of medium (DMEM supplemented with 10% fetal bovine serum (Gibco), 1% penicillin, streptomycin and L- glutamine). The cells are incubated overnight at 37°C in an atmosphere of 5% CO 2 .
- Compounds are added the next day to a final concentration of 25 ⁇ M along with 1 mM aminoguanidine (inhibits serum amine oxidase) and 2.5 mM DFMO in a final volume of 200 ⁇ L medium.
- the positive control contained 25 ⁇ M spermidine.
- the cells are allowed to incubate for 6 days before cell growth is measured by MTS/PMS dye assay (Cell Titer 96 Aqueous Non- Radioactive Cell Proliferation Assay, Promega).
- the rescue coefficients in Fig. 1 and Fig. 5 are expressed as the ratio of the O.D. ((compound + 2.5 mM DFMO)/O.D. (2.5 mM DFMO alone)).
- DFMO alone has a value of one. If the drag has the desired effect of no rescue then it has a value close to DFMO alone (i.e. about 1). If the value is less than one, then the compound is growth inhibitory. If the value is 0, the compound is cytotoxic. Compounds that rescued cells from DFMO induced growth inhibition give ratios higher than one and are less desirable as therapeutics. As shown in Fig 5, neither compound A nor B significantly rescue cells from DFMO- induced growth inhibition. Compound A is the least effective in comparison to the spermidine control. Agmatine is somewhat better than either A or B in rescuing cells from DFMO induced growth inhibition.
- a useful factor to compare the effectiveness of the various antizyme frameshifters takes into account their potency for inducing frameshifting and inability to rescue cells from DFMO- induced growth inhibition. This measure is referred to as the frameshift-rescue factor (FRF).
- FRF is calculated by multipling the %RF value by the inverse of the rescue coefficient. This method greatly increases the simplicity of analyzing multiple data parameters. If either the %RF value is low or the rescue coefficient high, the compound does not stand out as a potential candidate. Using this analysis with compounds A, B and agmatine, FRF values of 120, 83 and 17 are obtained, respectively. Based on this method of analysis, compound A potentially has the greatest ability to deplete polyamine levels and inhibit cell growth.
- EXAMPLE 8 Depletion of Intracellular Polyamine Levels with Compound A
- Compound A is further evaluated to determine if it depletes intracellular polyamine levels in HEK-293 cells in a dose-dependent fashion (Fig. 6).
- HEK-293 cells are plated in 75 cm 2 flasks at 300,000 cells/mL to insure they would be in log growth for the duration of the experiment. After overnight incubation at 37°C in an atmosphere of 5% CO , compound A is added along with 1 mM aminoguanidine. The flasks are then incubated for 6 days at 37°C in an atmosphere of 5% CO 2 .
- the cells are harvested by washing twice with ice-cold IX PBS, trypsinized, counted, and lysed in 0.4 N perchloric acid. Diaminoheptane is used in all dansylation reactions as an internal standard. Peak area is normalized by dividing the peak area of the compound divided by the peak area of the internal standard.
- the HPLC method for the fluorometric detection of polyamines from the cell extracts is based on the procedure by Kabra (Kabra, P. M. et. al. J. Chromatogr. 380:19-32 (1986)). It is expected that if compound A induces frameshifting and therefore elevates the level of antizyme, intracellular levels of polyamines would decrease. This is observed in HEK-293 cells, which show significant reductions of intracellular levels of polyamines (Fig. 6). Putrescine is most sensitive to the treatment and is non- detectable at the lowest concentration tested.
- Spemidine levels are down by about 90%o at 10 ⁇ M and are non-detectable when incubated with 100 ⁇ M of compound A. Cell growth is also affected, decreasing by 32% at 10 ⁇ M and 38.5% at 30 ⁇ M.
- the intracellular levels of compound A also increase dose-dependently (Fig.
- Medi Quest Therapeutics have found that agmatine inhibits growth with an IC 50 of approximately 2 mM in MDA-MB-231 cells, 5 mM in the prostate PC-3 cells line and 0.21 mM in HEK-293 cells (data not shown). Both compound A and B are found to be more potent than agmatine in inducing antizyme frameshifting (Fig 2). Compound A is also a more potent inhibitor of HEK- 293 cell growth with an IC 50 of 60 ⁇ M and is cytotoxic at 1 mM. It has been previously shown that forced expression of antizyme can result in cell death. The toxicity of compound A observed at 1 mM suggests that a potent antizyme inducer can be cytotoxic when the antizyme levels reach a sufficiently high threshold value.
- l,4-bis[bis(3-aminopropyl) xylene diamine can enter the cell and inhibit transport at least in part through induction of full length antizyme.
- the pharmaceutically acceptable carriers described herein for example, vehicles, adjuvants, excipients, or diluents, are well-known to those who are skilled in the art.
- the pharmaceutically acceptable carrier is chemically inert to the active compounds and has no detrimental side effects or toxicity under the conditions of use.
- the pharmaceutically acceptable carriers can include polymers and polymer matrices.
- the compounds of this invention can be administered by any conventional method available for use in conjunction with pharmaceuticals, either as individual therapeutic agents or in a combination of therapeutic agents.
- the dosage administered well vary depending upon known factors, such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the age, health and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment; the frequency of treatment; and the effect desired.
- a daily dosage of active ingredient can be expected to be about 0.001 to 1000 milligrams (mg) per kilogram (kg) of body weight, with the preferred dose being 0.1 to about 30 mg/kg.
- Dosage forms contain from about 1 mg to about 500 mg of active ingredient per unit.
- the active ingredient will ordinarily be present in an amount of about 0.5-95% weight based on the total weight of the composition.
- the active ingredient can be administered orally in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups and suspensions. It can also be administered parenterally, in sterile liquid dosage forms.
- the active ingredient can also be administered intranasally (nose drops) or by inhalation of a drag powder mist.
- Other dosage forms are potentially possible such as administration transdermally, via patch mechanism or ointment.
- Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, saline, or orange juice; (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, as solids or granules; (c) powders; (d) suspensions in an appropriate liquid; and (e) suitable emulsions.
- Liquid formulations may include diluents, such as water and alcohols, for example, ethanol, benzyl alcohol, propylene glycol, glycerin, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agent, or emulsifying agent.
- diluents such as water and alcohols, for example, ethanol, benzyl alcohol, propylene glycol, glycerin, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agent, or emulsifying agent.
- Capsule forms can be of the ordinary hard- or soft- shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and corn starch.
- Tablet forms can include one or more of the following: lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible carriers.
- Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acadia, emulsions, and gels containing, in addition to the active ingredient, such carriers as are known in the art.
- the compounds of the present invention alone or in combination with other suitable components, can be made into aerosol formulations to be administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, and nitrogen.
- Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aq ⁇ eous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
- the compound can be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol such as poly(ethyleneglycol) 400, glycerol ketals, such as 2,2-dimethyl-l,3-dioxolane-4-methanol, ethers, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adj
- Oils which can be used in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.
- Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts
- suitable detergents include (a) cationic detergents such as, for example, dimethyldialkylammonium halides, and alkylpyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefm sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylene polypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl ⁇ -aminopropionates, and 2- alkylimidazoline quaternary ammonium salts, and (e) mixtures thereof.
- cationic detergents such as,
- the parenteral formulations typically contain from about 0.5% to about 25% by weight of the active ingredient in solution. Suitable preservatives and buffers can be used in such formulations. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations ranges from about 5% to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethyl ene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol.
- HLB hydrophile-lipophile balance
- compositions of the present invention are also well-known to those who are skilled in the art. The choice of excipient will be determined in part by the particular compound, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the pharmaceutical composition of the present invention. The following methods and excipients are merely exemplary and are in no way limiting.
- the pharmaceutically acceptable excipients preferably do not interfere with the action of the active ingredients and do not cause adverse side-effects.
- Suitable carriers and excipients include solvents such as water, alcohol, and propylene glycol, solid absorbants and diluents, surface active agents, suspending agent, tableting binders, lubricants, flavors, and coloring agents.
- the formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use.
- sterile liquid excipient for example, water
- Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.
- the requirements for effective pharmaceutical carriers for injectable compositions are well known to those of ordinary skill in the art. See Pharmaceutics and
- Formulations suitable for topical administration include lozenges comprising the active ingredient in a flavor, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia; and mouthishes comprising the active ingredient in a suitable liquid carrier; as well as creams, emulsions, and gels containing, in addition to the active ingredient, such carriers as are known in the art.
- formulations suitable for rectal administration may be presented as suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases.
- Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field.
- the dose administered to an animal, particularly a human, in the context of the present invention should be sufficient to effect a therapeutic response in the animal over a reasonable time frame.
- dosage will depend upon a variety of factors including a condition of the animal, the body weight of the animal, as well as the condition being treated.
- a suitable dose is that which will result in a concentration of the active agent in a patient which is known to effect the desired response.
- the size of the dose also will be determined by the route, timing and frequency of administration as well as the existence, nature, and extend of any adverse side effects that might accompany the administration of the compound and the desired physiological effect.
- Hard Shell Capsules A large number of unit capsules are prepared by filling standard two-piece hard gelatine capsules each with 100 mg of powdered active ingredient, 150 mg of lactose, 50 mg of cellulose and 6 mg of magnesium stearate.
- Soft Gelatin Capsules A mixture of active ingredient in a digestible oil such as soybean oil, cottonseed oil or olive oil is prepared and injected by means of a positive displacement pump into molten gelatin to form soft gelatin capsules containing 100 mg of the active ingredient. The capsules are ished and dried.
- the active ingredient can be dissolved in a mixture of polyethylene glycol, glycerin and sorbitol to prepare a water miscible medicine mix.
- Tablets A large number of tablets are prepared by conventional procedures so that the dosage unit is 100 mg of active ingredient, 0.2 mg. of colloidal silicon dioxide, 5 mg of magnesium stearate, 275 mg of microcrystalline cellulose, 11 mg. of starch, and 98.8 mg of lactose. Appropriate aqueous and non-aqueous coatings may be applied to increase palatability, improve elegance and stability or delay absorption. Immediate Release Tablets/Capsules These are solid oral dosage forms made by conventional and novel processes. These units are taken orally without water for immediate dissolution and delivery of the medication.
- the active ingredient is mixed in a liquid containing ingredient such as sugar, gelatin, pectin and sweeteners. These liquids are solidified into solid tablets or caplets by freeze drying and solid state extraction techniques.
- the drug compounds may be compressed with viscoelastic and thermoelastic sugars and polymers or effervescent components to produce porous matrices intended for immediate release, without the need of water.
- the compounds of the present invention can be administered in the form of nose drops, or metered dose and a nasal or buccal inhaler.
- the drug is delivered from a nasal solution as a fine mist or from a powder as an aerosol.
Abstract
Description
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---|---|---|---|---|
WO2000046187A2 (en) * | 1999-02-05 | 2000-08-10 | Oridigm Corporation | Antizyme modulators and their use |
US6914079B2 (en) * | 2002-09-23 | 2005-07-05 | Mediquest Therapeutics, Inc. | Polyamine analogs that activate antizyme frameshifting |
US7144920B2 (en) * | 2002-09-23 | 2006-12-05 | Mediquest Therapeutics, Inc. | Polyamine analogs that activate antizyme frameshifting |
-
2004
- 2004-03-29 AU AU2004319105A patent/AU2004319105A1/en not_active Abandoned
- 2004-03-29 EP EP04821940A patent/EP1730100A1/en not_active Withdrawn
- 2004-03-29 WO PCT/US2004/009582 patent/WO2005105729A1/en not_active Application Discontinuation
- 2004-03-29 CA CA002555862A patent/CA2555862A1/en not_active Abandoned
Non-Patent Citations (1)
Title |
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See references of WO2005105729A1 * |
Also Published As
Publication number | Publication date |
---|---|
WO2005105729A1 (en) | 2005-11-10 |
CA2555862A1 (en) | 2005-11-10 |
AU2004319105A1 (en) | 2005-11-10 |
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