WO1998025596A2 - Use of a 6-aryl pyrimidine compound for treating multiple sclerosis - Google Patents
Use of a 6-aryl pyrimidine compound for treating multiple sclerosis Download PDFInfo
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- WO1998025596A2 WO1998025596A2 PCT/US1997/021402 US9721402W WO9825596A2 WO 1998025596 A2 WO1998025596 A2 WO 1998025596A2 US 9721402 W US9721402 W US 9721402W WO 9825596 A2 WO9825596 A2 WO 9825596A2
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- amino
- pyrimidinol
- bromo
- iodo
- chloro
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- 0 Cc1c(*)c(*)nc(C)n1 Chemical compound Cc1c(*)c(*)nc(C)n1 0.000 description 2
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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
- A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
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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
-
- 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 provides a new use of known compounds. More particularly the present invention provides a method of treating multiple sclerosis using a series of known pyrimidiols including bropirimine.
- US Patent 4,619,933 claims a process of treating aplastic anemia with 2- amino-6-aryl-4-pyrimidinol compounds and discloses that alteration of the immunoregulatory system of the host animal and that administration of the active compounds increases antibody formation and be used to treat acquired or congenital hypogammaglobulinemia; activates macorphages and can be used to treat or prevent intracellular or extracellular parasitic infections, including bacterial and protozoal; increases hematopoietic stem cells in bone marrow and spleen and can be used to treat or prevent aplastic anemia; and decreases generation of allospecific killer cells and can be used to prevent rejection of organ and skin grafts.
- US Patent 5,434,157 claims various 6-aryl pyrimidinol compounds and compositions thereof as well a method for treating viral infections and inducing interferon production.
- this patent also discloses that the compounds have immunoregulatory activity; for example the compounds increased antibody formation and decreased delayed hypersensitivity and are useful in the treatment of parasitic diseases, organ transplants and skin graft rejections, and immune deficiencies including those caused as a side effect of therapy with cytotoxic agents and radiation.
- 08/419,963 claims the process of treating susceptible forms of cancer with 2-amino-6-aryl-4-pyrimidinol compounds and discloses the alteration of the immunoregulatory system of the host animal and that administration of the active compounds increases antibody formation and be used to treat acquired or congenital hypogammaglobulinemia; increases natural killer cells and can be used to treat various forms of cancer; activates macorphages and can be used to treat or prevent intracellular and can be used to treat or prevent intracellular or extracellular parasitic infections, including bacterial and protozoal; increases hematopoietic stem cells in bone marrow and spleen and can be used to treat or prevent aplastic anemia; and decreases generation of allospecific killer cells and can be used to prevent rejection of organ and skin grafts.
- Bropirimine (2-amino-5-bromo-6-phenyl-4(lH)-pyrimidinone; ABPP) which was selected from this group of 6-arylpyrimidinones for further development, is an orally active inducer of endogenous interferon, with established antiviral, immunostimulatory and antitumor activity. Regulatory approval from the United States Food and Drug Administration is being sought to market bropirimine tablets for the treatment of superficial bladder cancer (CIS).
- CIS superficial bladder cancer
- MMPIs matrix metalloproteinase inhibitors
- MS multiple sclerosis
- MMPIs matrix metalloproteinase inhibitors
- lymphokines such as interferon-beta
- Swanborg, R.H. Experimental Autoimmune Encephalomyelitis in Rodents as a Model for Human Demyelinating Disease. Clin. Immunol. Immunopath.
- interferons dosing of the interferons is restricted to parenteral injections, which must be repeated regularly, and there are indications that the administration of the interferons is unpleasant enough to result in a relatively high dropout rate for patients who initially attempt to use this therapy. Furthermore, the efficacy reported is weak enough to warrant continuing the search for better therapy to treat the disease.
- One concern associated with the direct use of interferons is the failure of the natural regulation systems to be able to control the concentrations of the cytokine. Since lymphokine/cytokine systems are usually tightly regulated, such a lack of internal control systems may result in side effects and less than optimal efficacy.
- Toxicological problems associated with interferons include neurological problems, such as changes in electroencephalograms, sleep patterns and cognition, and a flu-like syndrome [Spiegel, R.J., The alpha interferons: Clinical overview. Seminars in Oncology 14: 1-12 (1987) and Rohatiner, A.Z.S., Prior, P.F., and Burton, A.C., Central nervous system toxicity of interferon. Br. J. Cancer 47: 419-422 (1983)]. Furthermore, the full benefit of the cytokine may not be realized because the exogenous interferon concentration is determined strictly by timing of the doses and the dose administered. The role of naturally occurring interferon in MS is not understood.
- EAE Experimental autoimmune encephalomyelitis
- EAE in mice is often accompanied by demyelination in addition to the perivascular mononuclear cell infiltrates in the CNS.
- perivascular mononuclear cell infiltrates are readily observed in EAE, but the lesions are not usually accompanied by demyelination.
- the use of the animal models has significantly contributed to our understanding of some of the cellular events critical for initiating the disease. For example, a transient form of EAE can be transmitted between animals by transferring appropriately selected CD4 + T lymphocytes. This indicates a key role for the CD4 + T cells.
- results from the animal models have shown that particular amino acid sequences from MBP and proteolipid protein (PLP), which are the major protein constituents of myelin, may be key antigenic determinants in initiation and/or progression of EAE.
- PBP proteolipid protein
- the importance of the immunoreactivity to these proteins has subsequently been confirmed in patients with MS.
- the genetics associated with susceptibility to disease and the biochemical consequences of gene expression may be studied in considerably greater detail using animal models than may be carried out in human populations, especially by making use of a number of inbred strains of mice.
- the origin of the macrophage-like cells appears to be a combination of cells from the periphery and differentiated microglial cells [Huitinga, I., Ruuls, S.R., Jung, S., Van Rooijen, N., Hartung, H.-P., and Dijkstra, CD., Macrophages in T Cell Line-Mediated, Demyelinating, and Chronic Relapsing
- Suppressor cells also appear to play an important role in progression and control of EAE [Sun, D., Qin, Y., Chluba, J., Epplen, J.T., and Wekerle, H., Suppression of Experimentally Induced Autoimmune Encephalomyelitis by Cytolytic T-T Cell Interactions. Nature 332: 843-845 (1988)], although the particular type of cell involved in the suppression activity is only beginning to emerge [Fabry, Z., Raine, C.S., and Hart, M.N., Nervous tissue as an immune compartment: the dialect of the immune response in the CNS. Immunol. Today 15: 218-224 (1994)].
- This invention relates to the use of 6-aryl pyrimidine compounds for the treatment of multiple sclerosis (MS).
- the present invention a method of treating multiple sclerosis (MS) comprising the systemic administration of an effective amount of a compound represented by the formula
- Xg is equal to X, X 4 or X 5 wherein X 4 is fluoro, chloro, bromo or iodo, and X 5 is mono-, di or trihalomethyl, mono, di or trifluoroethyl, perfluoropropyl, and wherein X is alkyl of from 1 to 3 carbon atoms, inclusive, including isomeric forms, 2 propynyl and 2-propenyl, and X ⁇ is a member selected from the group consisting of
- R, R ⁇ , R2, R3, R4 is not hydrogen and wherein R or R4 is alkyl of from 1 to 8 carbon atoms, inclusive, including isomeric forms, alkoxy of from 1 to 8 carbon atoms, inclusive, including isomeric forms, fluoro, chloro, bromo, iodo or nitro; R- ⁇ or Rg is fluoro, chloro, bromo, iodo, nitro; trifluoromethyl or alkoxy of from 1 to 8 carbon atoms, inclusive, including isomeric forms, alkoxyethyloxy wherein alkoxy is from 1 to 5 carbon atoms, inclusive, including isomeric forms, or
- Rg and Rg are the same or different and are alkyl of from 1 to 8 carbon atoms, inclusive, including isomeric forms, benzyl, or taken together with -N> are a saturated cycloalkylamino group
- n' is 3, 4, 5 or 6 or dialkyl substituted cycloalkylamino wherein each alkyl is from 1 to 3 carbon atoms, inclusive; including isomeric forms, and R2 is chloro, fluoro, bromo, iodo, or alkyl of from 1 to 3 carbon atoms, inclusive;
- R, R ⁇ , R 2 , 3 and R 4 are not hydrogen and are the same or different and are fluoro, chloro, bromo, iodo, alkyl of from 1 to 8 carbon atoms, inclusive,including isomeric forms, alkoxy of from 1 to 8 carbon atoms, inclusive, including isomeric forms, nitro and trifluoromethyl;
- R 7 is substituted in either ring and is hydrogen, alkyl of from 1 to 8 carbon atoms, inclusive, including isomeric forms, alkoxy of from 1 to 8 carbon atoms, inclusive, including isomeric forms, fluoro, chloro, iodo, bromo or nitro- provided that when Xg is X, X ⁇ is not ⁇ -napthyl; (f) 2-furyl,
- Preferred compounds are: 2-amino-5-iodo-6-(3-bromophenyl)-4-pyrimidinol; 2-amino-5-bromo-6-(3-fluorophenyl)-4-pyrimidinol; 2-amino-5-bromo-6-(3-ethoxyethylphenyl)-4-pyrimidinol; 2-amino-5-bromo-6-(2-methoxyphenyl)-4-pyrimidinol; 2-amino-5-chloro-6-(2-methoxyphenyl)-4-pyrimidinol; 2-amino-5-iodo-6-(2-methoxyphenyl)-4-pyrimidinol;
- Bropirimine (2-amino-5-bromo-6-phenyl-4(lH)-pyrimidinone; ABPP) is particularly preferred and exists as an odorless white crystalline powder which melts with decomposition near 270 C.
- Bropirimine exists in several crystal forms comprising two general classes. One form, the hydroscopic, can undergo transition to at least two other polymorphic crystal forms; the other is a nonhydroscopic form stable to polymorphic transition. At 100% relative humidity, the hydroscopic form of bropirimine gains about 6.3% water, which is equivalent to the amount of water required to form a monohydrate. The nonhydroscopic drug does not show similar behavior and is preferred.
- 6-aryl pyrimidine compounds used in the present invention can be prepared by the general procedure(s) disclosed therein.
- the hydroscopic crystal form of bropirimine can be synthesized by treating 2- amino-6-phenyl-4(3H)-pyrimidine with bromine in acetic acid. The material is filtered from the mixture and dissolved in aqueous base. The material is reprecipitated by neutralization with acetic acid, filtered, washed with water, and dried.
- the non-hydroscopic crystal form of bropirimine can be obtained by heating an aqueous suspension of the hydroscopic crystal form above 70° C. The material is filtered, washed with water, and dried.
- Part A A mixture of guanidine carbonate, (39.4 g) n-butyl alcohol, and ethyl benzoylacetate (60 g) is reacted at or above 90°C The reaction is monitored by HPLC until the reaction is judged to be complete. The mixture is cooled and then acetic acid is added to pH 5.8-8.0 to complete the crystallization. The slurry is cooled further (0-5°C) and then the slurry is filtered. The cake is washed with n- butyl alcohol, and then with water. The cake of 2-amino-6-phenyl-4(3H)-pyrimidine may or may not be dried.
- Part B Liquid bromine (about 41.6 g) is added slowly to a slurry of 2-amino- 6-phenyl-4(3H)-pyrimidine (about 45.0 g on a dry basis) in acetic acid (273.7 ml) and water (273.0 ml) until the reaction is judged to be complete by HPLC. The reaction is quenched with the addition of 50% sodium hydroxide (36.2 g). The reaction mixture is heated to 75-90°C for 1 or more hours. The slurry is cooled and filtered. The cake is washed with water and dried to yield the nonhydroscopic crystal form of bropirimine.
- the mode of administration of the compounds of Formula I can be by the means disclosed in US Patent 5,434,157, preferably orally.
- the dosage administered will be dependent upon the condition of the patient, its age, health, weight, kind of concurrent treatment, if any, frequency of treatment, therapeutic ratio, and tolerance.
- the timing of drug administration can be important in maintenance of activity in the treatment of multiple sclerosis.
- dosage levels of the administered compounds of Formula I can be intravenous, 0.1 to about 50 mg/kg.; subcutaneous, 0.1 to about 150 mg./kg.; intramuscular, 0.1 to about 150 mg/kg.; and about 0.1 to about 400 mg/kg orally, preferably 15 to about 150 mg/kg of animal (body) weight.
- the active ingredients to be employed in the treatment of MS can be easily prepared in unit dosage form with the employment of pharmaceutical materials as disclosed in US Patent 5,434,157.
- the active ingredients are administered, in divided doses, of about 50 to about 1,000 mg kg/week of human body weight.
- a preferred oral dosage regimen of the present invention is the administration of three grams per day (divided over a six hour period) for three consecutive days per week, e.g. one gram of the active ingredient (preferably presented in unit dosage form) every two hours for three doses on days 1, 2 and 3 of each week for about three to about twelve months, or longer.
- An alternative preferred oral dosage regimen is the administration of three grams of the active ingredient (preferably presented in unit dosage form) per day (divided over a six hour period) every other day for up to about one year, or longer.
- Bropirimine which is representative of the compounds of Formula I, has been evaluated in an EAE model (Procedure A) and representative results shown in Figures 1-3.
- Figure 1 Effect of ABPP on EAE in mice. Mice were dosed with 250 milligrams per kilogram (mpk) on the days indicated by arrows. Two priming doses were administered on consecutive days, and only these doses were given intraperitoneally (i.p.) into mice. Subsequent doses, all were administered orally via a gavage tube followed at 3 or 4 day intervals to result in a twice-a-week dosing regimen. The mean scores for 40 mice in each group were statistically significantly less in ABPP treated mice beginning at day 11 and continued through day 29, that is 7 days after dosing was stopped during the first dosing interval. No significant decline in disease scores was detected during the second dosing interval.
- mpk milligrams per kilogram
- Figure 2 Effect of two different doses of ABPP on EAE in mice. All doses of ABPP were administered orally using a gavage tube. Two priming doses on days 7 and 8 were followed with doses given twice a week for a total of three weeks.
- the mean disease in mice treated with 400 milligrams per kilogram (mpk) ABPP was statistically significantly less than in mice treated with vehicle alone or with 100 mpk.
- the mean disease was statistically highly significantly lower for the group treated with 400 mpk beginning on approximately day 15 and continuing through day 37, i.e. approximately 1 week after dosing was stopped.
- the mean disease for mice treated with 100 mpk ABPP was statistically significantly less than in the vehicle-treated group only on days 16 and 17.
- Each group began with 65 mice per group, and 5 mice were removed at three times during the course of the experiment for histological evaluation of the lower spinal cords. Additionally, 5 mice from each group were processed for histology on days 43 and 56.
- FIG. 3 Dose response for ABPP.
- Two priming doses of ABPP were given via gavage intubation on days 6 and 7 after injection of MBP to initiate EAE. Dosing was continued at 3 day intervals through day 25. The days that doses were administered to the mice are indicated by arrows just above the abscissa on the graph. Approximately equivalent and statistically significant suppression of disease was observed beginning on day 6 for all 3 doses of ABPP used. After day 16, the degree of disease suppression was greatest in mice treated with 400 mpk ABPP, and the mice treated with 400 mpk had significantly less disease than the mice treated with either 100 mpk or 200 mpk. However, it was difficult to distinguish between the degree of effect of the lower two doses.
- mice (Jackson Laboratories, Bar Harbor, Maine) are injected intramuscularly in a rear flank on day 0 with 150 ⁇ g of purified guinea pig myelin basic protein (MBP) in complete Freund's adjuvant supplemented with 2.5 mg/ml M. tuberculosis.
- MBP myelin basic protein
- B. pertussis vaccine (Michigan Dept. of Health) is injected subcutaneously at the base of the tail.
- a second dose of B. pertussis is injected two days later, on day 3.
- the first signs of disease are usually observed in a few mice beginning at day 6-7 and the mean day of disease onset is approximately day 10.
- the mice are scored approximately 6 times per week for changes in motor activity according to the following scale:
- Dosing for the test compound is via a gavage tube inserted into the stomach of a mouse, unless indicated otherwise.
- the volume administered is 0.5 ml.
- the vehicle used with bropirimine (ABPP) contained 5.26 mg carboxymethylcellulose, 4.0 mg polysorbate 80 NF, 9.0 mg sodium chloride, and 9.18 mg benzylalcohol NF.
- mice are perfused with formalin to fix tissues in situ.
- Eight vertebrae, thoracic 10-13 and lumbar 1-4, are removed from the carcass, further fixed in formalin, decalcified using a 2 x 4 hr incubation in formic acid, carefully separated into individual vertebra and placed in cassettes for paraffin embedding, sectioning, and staining with hematoxylin and eosin (H&E).
- H&E hematoxylin and eosin
- Five, 5 ⁇ m thick spinal cord cross sections are cut, each 100 microns apart, from each of the 8 vertebrae for a total of 40 cross sections per mouse.
- the cross sections are scored for the presence and degree of infiltration. For an individual mouse, the results are presented as the proportion of cross sections with leukocyte infiltration, regardless of the degree of infiltration, or as the sum of the histological scores for all 40 cross sections. Histology samples were scored according to the following scale: Score
- Murine interferon determinations are performed using a modification of the method published previously [Stringfellow, D.A., Vanderberg, H.C, Weed, S.D., Interferon induction by 5-halo-6-phenyl pyrimidinones. J. Interferon Res. 1:1-14 (1980)].
- L929 (ATCC) cells are used at 90% confluence in 24 well plates. Samples for interferon determination are diluted into DMEM (Gibco) tissue culture medium containing 10% fetal bovine sera and antibiotics. Dilutions of samples are then added to wells of L929 cells and the cells became 100% confluent overnight.
- DMEM Gibco
- VSV vesicular stomatitis virus
- Virus is allowed to adsorb to cells for one hour, and the wells are overlaid with 0.9% agarose prepared in tissue culture medium containing 2% sera. Cell cultures are incubated for 40-44 hrs, and VSV plaques are counted after 3 hrs of staining with neutral red. Included in each interferon assay are samples of interferon standards obtained from the NIAID Repository, NIH. The standards included murine alpha interferon
- Figure 4 Interferon concentrations in peripheral blood in vehicle- and ABPP treated mice. Blood samples were collected on the days indicated and assessed for interferon concentration. Results shown correspond to the experiment shown in Figure 2.
- Figure 5. Interferon concentrations in peripheral blood in vehicle- and ABPP treated mice. Blood samples were collected on the days indicated and assessed for interferon concentration. Results shown correspond to the experiment shown in Figure 3.
Abstract
A method for treating multiple sclerosis by systemic administration of a 6-aryl pyrimidine compound or a pharmaceutically acceptable salt thereof in association with a pharmaceutical carrier to a human having symptoms of multiple sclerosis.
Description
METHOD FOR TREATING MULTIPLE SCLEROSIS BACKGROUND OF THE INVENTION
The present invention provides a new use of known compounds. More particularly the present invention provides a method of treating multiple sclerosis using a series of known pyrimidiols including bropirimine.
The preparation and use of 2-amino-5-halo-6-alkyl-4-pyrimidiols as antiviral agents is known US Patent 3,956,302 and Nicols, Weed and Underwood, Antimicrobial Agents, Chemo. Ther. 9 433, 1976. The preparation and use of 2-amino-6-aryl-4-pyrimidinol compounds for alteration of the immunoregulatory system of the host animal is disclosed in US Patent 4,543,248. US Patent 4,543,248 claims a process for treating acquired or congenital hypogammaglobulinemia, acquired or congenital agammaglobulinemia with 2-amino-6-aryl-4-pyrimidinol compounds. US Patent 4,507,302 discloses that the immunoregulatory system is altered or arthritis treated by systemic administration of 2-amino-6-aryl-4-pyrimidinol compounds.
US Patent 4,619,933 claims a process of treating aplastic anemia with 2- amino-6-aryl-4-pyrimidinol compounds and discloses that alteration of the immunoregulatory system of the host animal and that administration of the active compounds increases antibody formation and be used to treat acquired or congenital hypogammaglobulinemia; activates macorphages and can be used to treat or prevent intracellular or extracellular parasitic infections, including bacterial and protozoal; increases hematopoietic stem cells in bone marrow and spleen and can be used to treat or prevent aplastic anemia; and decreases generation of allospecific killer cells and can be used to prevent rejection of organ and skin grafts.
US Patent 5,002,951 claims a method for treating bacterial and protozoal infections with 2-amino-6-aryl-4-pyrimidinol compounds.
US Patent 5,434,157 claims various 6-aryl pyrimidinol compounds and compositions thereof as well a method for treating viral infections and inducing interferon production. In addition, this patent also discloses that the compounds have immunoregulatory activity; for example the compounds increased antibody formation and decreased delayed hypersensitivity and are useful in the treatment of parasitic diseases, organ transplants and skin graft rejections, and immune deficiencies including those caused as a side effect of therapy with cytotoxic agents and radiation.
US Patent Application Serial No. 08/419,963 claims the process of treating susceptible forms of cancer with 2-amino-6-aryl-4-pyrimidinol compounds and discloses the alteration of the immunoregulatory system of the host animal and that administration of the active compounds increases antibody formation and be used to treat acquired or congenital hypogammaglobulinemia; increases natural killer cells and can be used to treat various forms of cancer; activates macorphages and can be used to treat or prevent intracellular and can be used to treat or prevent intracellular or extracellular parasitic infections, including bacterial and protozoal; increases hematopoietic stem cells in bone marrow and spleen and can be used to treat or prevent aplastic anemia; and decreases generation of allospecific killer cells and can be used to prevent rejection of organ and skin grafts.
Bropirimine (2-amino-5-bromo-6-phenyl-4(lH)-pyrimidinone; ABPP) which was selected from this group of 6-arylpyrimidinones for further development, is an orally active inducer of endogenous interferon, with established antiviral, immunostimulatory and antitumor activity. Regulatory approval from the United States Food and Drug Administration is being sought to market bropirimine tablets for the treatment of superficial bladder cancer (CIS).
A variety of compounds have been proposed for use in the treatment of multiple sclerosis (MS) including matrix metalloproteinase inhibitors (MMPIs) [Gijbels, K.,Galardy, R.E., Steinman, L., Reversal of Experimental Autoimmune Encephalomyelitis with a Hydroxamate Inhibitor of Matrix Metalloproteinases. [J. Clin. Invest. 94: 2177-2182 (1994)], lymphokines, such as interferon-beta [Swanborg, R.H., Experimental Autoimmune Encephalomyelitis in Rodents as a Model for Human Demyelinating Disease. Clin. Immunol. Immunopath. 77: 4-13 (1995) and Yu, M., Nishiyama, A., Trapp, B.D. and Tuohy, V.K., Interferon-beta Inhibits Progression of Relapsing-Remitting Experimental Autoimmune Encephalomyelitis. J. Neuroimmunol. 64: 91-100 (1996)], and antibodies against surface markers such as VLA-4 [Baron, J.L., Madri, J.A., Ruddle, N.H., Hashim, G., Janeway, Jr., C.A., Surface Expression ofAlpha-4 Integrin by CD4 T-cells is Required for their Entry into Brain Parenchyma. J. Exp. Med. 177: 57-68 (1993)]. Recently, linomide has entered clinical trials after demonstrated efficacy in the animal model of MS, experimental autoimmune encephalomyelitis (EAE) [Karussis, D.M., Lehmann, D., Slavin, S., Vourka-Karussis, U., Mizrachi-Koll, R., Ovadia, H., Kalland, T., and Abramsky, O., Treatment of Chronic-Relapsing Experimental Autoimmune Encephalomyelitis with the Synthetic Immunomodulator Linomide (quinoline-3-carboximide) . Proc. Natl. Acad. Sci. 90: 6400-6404 (1993) and
Karussis, D.M., Lehmann, D., Slavin, S., Vourka-Karussis, U., Mizrachi-Koll, R., Ovadia, H., Ben-Nun, A. Kalland, T., and Abramsky, O., Inhibition of acute, Experimental Autoimmune Encephalomyelitis by the Synthetic Immunomodulator Linomide. Ann. Neurol. 34: 654-660 (1993)]. The most recently approved biologicals for the treatment of MS are from the cytokine/lymphokine family, i.e. β-interferons. However, dosing of the interferons is restricted to parenteral injections, which must be repeated regularly, and there are indications that the administration of the interferons is unpleasant enough to result in a relatively high dropout rate for patients who initially attempt to use this therapy. Furthermore, the efficacy reported is weak enough to warrant continuing the search for better therapy to treat the disease. One concern associated with the direct use of interferons is the failure of the natural regulation systems to be able to control the concentrations of the cytokine. Since lymphokine/cytokine systems are usually tightly regulated, such a lack of internal control systems may result in side effects and less than optimal efficacy. Toxicological problems associated with interferons include neurological problems, such as changes in electroencephalograms, sleep patterns and cognition, and a flu-like syndrome [Spiegel, R.J., The alpha interferons: Clinical overview. Seminars in Oncology 14: 1-12 (1987) and Rohatiner, A.Z.S., Prior, P.F., and Burton, A.C., Central nervous system toxicity of interferon. Br. J. Cancer 47: 419-422 (1983)]. Furthermore, the full benefit of the cytokine may not be realized because the exogenous interferon concentration is determined strictly by timing of the doses and the dose administered. The role of naturally occurring interferon in MS is not understood. Experimental autoimmune encephalomyelitis (EAE) has been an accepted animal model of MS for many years. A review of the literature containing extensive citations was recently published comparing EAE and MS [Swanborg, R.H., Experimental autoimmune encephalomyelitis in rodents as a model for human demyelinating disease. Clin. Immunol. Immunopath. 77: 4-13 (1995)]. The most common models of EAE are those developed using rodents. Although EAE and MS are not identical, there are a number of important features in common between EAE and MS. For example, using mice, a chronic-remitting form of EAE, closely resembling the clinical appearance of MS in humans, has been developed. EAE in mice is often accompanied by demyelination in addition to the perivascular mononuclear cell infiltrates in the CNS. In contrast, in rats perivascular mononuclear cell infiltrates are readily observed in EAE, but the lesions are not usually accompanied by demyelination. Since the number and type of samples
obtained from human MS patients is necessarily limited, the use of the animal models has significantly contributed to our understanding of some of the cellular events critical for initiating the disease. For example, a transient form of EAE can be transmitted between animals by transferring appropriately selected CD4+ T lymphocytes. This indicates a key role for the CD4+ T cells. Furthermore, results from the animal models have shown that particular amino acid sequences from MBP and proteolipid protein (PLP), which are the major protein constituents of myelin, may be key antigenic determinants in initiation and/or progression of EAE. The importance of the immunoreactivity to these proteins has subsequently been confirmed in patients with MS. Furthermore, the genetics associated with susceptibility to disease and the biochemical consequences of gene expression may be studied in considerably greater detail using animal models than may be carried out in human populations, especially by making use of a number of inbred strains of mice. Another parallel between EAE and MS was recently demonstrated by showing that administration of interferon-beta to mice with EAE resulted in a decrease in symptoms and a decreased relapse rate [Yu, M., Nishiyama, A., Trapp, B.D. and Tuohy, V.K., Interferon-beta Inhibits Progression of Relapsing-Remitting Experimental Autoimmune Encephalomyelitis. J. Neuroimmunol. 64: 91-100 (1996)], similar to the results observed in MS patients. The cells comprising a lesion have been studied in both EAE and in MS.
Conclusions about the sequence of cell types that infiltrate lesions in the brain or spinal cord vary somewhat from author to author, but the early infiltration of CD4+ T-lymphocytes is well established [Fabry, Z., Raine, C.S., and Hart, M.N. , Nervous Tissue as an Immune Compartment: the Dialect of the Immune Response in the CNS. Immunol. Today 15: 218-224 (1994)]. At a later stage of disease, CD8+ T cells infiltrate lesions. In most demyelinating lesions, macrophage-like cells are present in large numbers. The origin of the macrophage-like cells appears to be a combination of cells from the periphery and differentiated microglial cells [Huitinga, I., Ruuls, S.R., Jung, S., Van Rooijen, N., Hartung, H.-P., and Dijkstra, CD., Macrophages in T Cell Line-Mediated, Demyelinating, and Chronic Relapsing
Experimental Autoimmune Encephalomyelitis in Lewis Rats. Clin. Exp. Immunol. 100: 344-351 (1995)] and Bauer, J., Huitinga, I., Zhao, W., Lassmann, H., Hickey, W., and Dijkstra, CD., The Role of Macrophages, Perivascular Cells, and Microglial Cells in the Pathogenesis of Experimental Autoimmune Encephalomyelitis. Glia 15: 437-446 (1995)]. Suppressor cells also appear to play an important role in progression and control of EAE [Sun, D., Qin, Y., Chluba, J., Epplen, J.T., and
Wekerle, H., Suppression of Experimentally Induced Autoimmune Encephalomyelitis by Cytolytic T-T Cell Interactions. Nature 332: 843-845 (1988)], although the particular type of cell involved in the suppression activity is only beginning to emerge [Fabry, Z., Raine, C.S., and Hart, M.N., Nervous tissue as an immune compartment: the dialect of the immune response in the CNS. Immunol. Today 15: 218-224 (1994)].
Following intraperitoneal (i.p.) or oral (p.o.) administration of bropirimine to mice, high interferon-alpha concentrations were observed in the serum and may have been produced predominantly in the spleen and thymus [Stringfellow, D.A. and Weed, S.D., δ-halo-6-phenyl pyrimidinones: A New Series of Interferon-Inducing Agents. In: Khan, A. Hill, N.O., Dorn, G.L., editors. Interferons: Properties and clinical uses. Proceedings International Symposium, Cleland Fikes Foundation Press, Dallas, TX 315-326 (1980) (Weed, S.D., Kramer, G.D., and Stringfellow, D.A. (1980) Antiviral Properties of 6-arylpyrimidinones . In: Nelson, J.D., and Grassi, C, editors. American Society Microbiology, 1980, 1408-1409; and Stringfellow, D.A., 6-Arylpyrimidinols: Interferon inducers-immuno-modulators- antiviral and antineoplastic agents. Prog. Cancer Res. Ther. 16: 215-228 (1981)]. 2',5'-oligoadenylate synthetase activity, which is induced by interferon, was also increased following bropirimine treatment of mice, consistent with interferon induction. Bropirimine induced production of IFN-alpha in vitro in cultured murine thymocytes and in bone marrow, and spleen cells [Stringfellow, D.A. and Weed, S.D., 5-halo-6-phenyl pyrimidinones: A New Series of Interferon-Inducing Agents. In: Khan, A. Hill, N.O., Dorn, G.L., editors. Interferons: Properties and clinical uses. Proceedings International Symposium, Cleland Fikes Foundation Press, Dallas, TX 315-326 (1980) and Hamilton, R.D., Buthala, D.A., Eidson, E.E., Tomilo, A., and Andrews, J.C., Interferon induction in vitro with 6-methyl and 6- aryl-pyrimidines. In: Nelson, J.D. and Grassi, C, editors. 11th Current Chemotherapy Infectious Disease, Proceedings International Congress Chemotherapy, American Society Microbiology, Washington (DC). 1980,2: 1409-1411]. IFN-alpha was induced by bropirimine in human tonsillar tissue
[Stringfellow, D.A., Vanderberg, H.C, Weed, S.D., Interferon Induction by δ-halo-6- phenyl Pyrimidinones. J. Interferon Res. 1:1-14 (1980)] and in peripheral mononuclear cells [Kita, M. and Imanishi, J., Induction of Interferon by Bropirimine in Human Peripheral Blood Mononuclear Cell Culture. Pharmacol. Ther. (Japan) 20: 37-42 (1992)]. Furthermore, IFN-alpha was detected in the sera of bropirimine-
treated patients. Induction of natural killer cell activity [Lotzova, E., Savary, C.A., and Stringfellow, D.A., 5-halo-6-phenyl-pyrimidinones; New molecules with cancer therapeutic potential and interferon-inducing capacity are strong inducers of murine natural killer cells. J. Immunol. 130: 965-969 (1983); Lotzova, E., Savary, C.A., Khan, A., and Stringfellow, D.A., Stimulation of Natural Killer Cells in Two Random-bred Strains of Athymic Rats by Interferon-Inducing Pyrimidinones. J. Immunol. 132: 2566-2570 (1984); and Lotzova, E., Savary, C.A., Lowlachi, M., and Murasko, D.M., Cytotoxic and Morphologic Profile of Endogenous and Pyrimidinone- Activated Murine NK Cells. J. Immunol. 136: 732-740 (1986)] and macrophage tumoricidal activity [Li, L., Wallace, T.L., Richard, K.A., and Tracey, D.E., Mechanisms of Antitumor Action of Pyrimidinones in the Treatment of B16 Melanoma and P388 Leukemia. Cancer Res. 45: 532-538 (1985)] have also been associated with bropirimine administration. Anti-inflammatory effects, typical of interferon-alpha inducers, have been demonstrated for bropirimine in chronic T cell mediated granuloma formation [Dunn. C.J., Galinet, L.A., Gibbons, A.J., and
Shields, S.K., Murine Delayed-Type Hypersensitivity Granuloma: An improved Model for the Identification and Evaluation of Different Classes of Anti-inflammatory Drugs. Int. J. Immunopharm. 12: 899-904 (1990)].
BRIEF DESCRIPTION OF THE INVENTION
This invention relates to the use of 6-aryl pyrimidine compounds for the treatment of multiple sclerosis (MS).
DETAILED DESCRIPTION OF THE INVENTION The present invention a method of treating multiple sclerosis (MS) comprising the systemic administration of an effective amount of a compound represented by the formula
HO
wherein Xg is equal to X, X4 or X5 wherein X4 is fluoro, chloro, bromo or iodo, and X5 is mono-, di or trihalomethyl, mono, di or trifluoroethyl, perfluoropropyl, and
wherein X is alkyl of from 1 to 3 carbon atoms, inclusive, including isomeric forms, 2 propynyl and 2-propenyl, and X^ is a member selected from the group consisting of
(a) phenyl,
(b) a morosubstituted phenyl of the formula
R5 / N
R6
wherein Rg and Rg are the same or different and are alkyl of from 1 to 8 carbon atoms, inclusive, including isomeric forms, benzyl, or taken together with -N> are a saturated cycloalkylamino group
wherein n' is 3, 4, 5 or 6 or dialkyl substituted cycloalkylamino wherein each alkyl is from 1 to 3 carbon atoms, inclusive; including isomeric forms, and R2 is chloro, fluoro, bromo, iodo, or alkyl of from 1 to 3 carbon atoms, inclusive;
(c) a disubstituted phenyl of the formula:
wherein any two of R, R^, R2, 3 and R4 are not hydrogen and are the same or different and are fluoro, chloro, bromo, iodo, alkyl of from 1 to 8 carbon atoms, inclusive,including isomeric forms, alkoxy of from 1 to 8 carbon atoms, inclusive, including isomeric forms, nitro and trifluoromethyl;
(d) a trihalo substituted phenyl wherein halo is chloro, bromo, iodo, or fluoro;
(e) ot-naphthyl of the formula:
wherein R7 is substituted in either ring and is hydrogen, alkyl of from 1 to 8 carbon atoms, inclusive, including isomeric forms, alkoxy of from 1 to 8 carbon atoms, inclusive, including isomeric forms, fluoro, chloro, iodo, bromo or nitro- provided that when Xg is X, X^ is not α-napthyl; (f) 2-furyl,
(g) 3-pyridyl,
(h) 2-pyridyl, and
(i) 2-pyrazyl, provided that when Xg is X, 2 propynyl or 2-propenyl, X^ is not 2-furyl, 3-pyridyl, 2-pyridyl or 2-pyrazyl, or a salt thereof, in association with a pharmaceutical carrier.
Preferred compounds are: 2-amino-5-iodo-6-(3-bromophenyl)-4-pyrimidinol; 2-amino-5-bromo-6-(3-fluorophenyl)-4-pyrimidinol; 2-amino-5-bromo-6-(3-ethoxyethylphenyl)-4-pyrimidinol; 2-amino-5-bromo-6-(2-methoxyphenyl)-4-pyrimidinol; 2-amino-5-chloro-6-(2-methoxyphenyl)-4-pyrimidinol;
2-amino-5-iodo-6-(2-methoxyphenyl)-4-pyrimidinol;
2-amino-5-bromo-6-(3-chlorophenyl)-4-pyrimidinol;
2-amino-5-iodo-6-(3-chlorophenyl)-4-pyrimidinol;
2-amino-5-chloro-6-(3-chlorophenyl)-4-pyrimidinol; 2-amino-5-chloro-6-(2-fluorophenyl)-4-pyrimidinol;
2-amino-5-chloro-6-(3-fluorophenyl)-4-pyrimidinol;
2-amino-5-bromo-6-(2-fluorophenyl)-4-pyrimidinol;
2-amino-5-iodo-6-(3-fluorophenyl)-4-pyrimidinol;
2-amino-5-iodo-6-phenyl-4-pyrimidinol; 2-amino-5-chloro-6-phenyl-4-pyrimidinol;
2-amino-5-bromo-6-phenyl-4-pyrimidinol;
2-amino-5-chloro-6-(3-methoxyphenyl)-4-pyrimidinol;
2-amino-5-bromo-6-(3-methoxyphenyl)-4-pyrimidinol;
2-amino-5-iodo-6-(3-methoxyphenyl)-4-pyrimidinol; 2-amino-5-bromo-6-(2-pyridyl)-4-pyrimidinol;
2-amino-5-iodo-6-(3,4-dichlorophenyl)-4-pyrimidinol;
2-amino-5-bromo-6-(α-naphthyl)-4-pyrimidinol;
2-amino-5-chloro-6-(3-nitrophenyl)-4-pyrimidinol;
2-amino-5-iodo-6-(3-nitrophenyl)-4-pyrimidinol; 2-amino-5-iodo-6-(3-trifluoromethylphenyl-4-pyrimidinol;
2-amino-5-ethyl-6-phenyl-4-pyrimidinol;
2-amino-5-bromo-6-(3,5-dimethoxyphenyl)-4-pyrimidinol and
2-amino-5-chloro-6-(3-propyloxyphenyl)-4-pyrimidinol.
Bropirimine (2-amino-5-bromo-6-phenyl-4(lH)-pyrimidinone; ABPP) is particularly preferred and exists as an odorless white crystalline powder which melts with decomposition near 270 C. Bropirimine exists in several crystal forms comprising two general classes. One form, the hydroscopic, can undergo transition to at least two other polymorphic crystal forms; the other is a nonhydroscopic form stable to polymorphic transition. At 100% relative humidity, the hydroscopic form of bropirimine gains about 6.3% water, which is equivalent to the amount of water required to form a monohydrate. The nonhydroscopic drug does not show similar behavior and is preferred.
Applicant hereby incorporates by reference, in their entirety, US Patents
5,434,157 and 4,619,933. The 6-aryl pyrimidine compounds used in the present invention can be prepared by the general procedure(s) disclosed therein.
The hydroscopic crystal form of bropirimine can be synthesized by treating 2-
amino-6-phenyl-4(3H)-pyrimidine with bromine in acetic acid. The material is filtered from the mixture and dissolved in aqueous base. The material is reprecipitated by neutralization with acetic acid, filtered, washed with water, and dried. The non-hydroscopic crystal form of bropirimine can be obtained by heating an aqueous suspension of the hydroscopic crystal form above 70° C. The material is filtered, washed with water, and dried.
Procedure 1: Preparation of Bropirimine
Part A: A mixture of guanidine carbonate, (39.4 g) n-butyl alcohol, and ethyl benzoylacetate (60 g) is reacted at or above 90°C The reaction is monitored by HPLC until the reaction is judged to be complete. The mixture is cooled and then acetic acid is added to pH 5.8-8.0 to complete the crystallization. The slurry is cooled further (0-5°C) and then the slurry is filtered. The cake is washed with n- butyl alcohol, and then with water. The cake of 2-amino-6-phenyl-4(3H)-pyrimidine may or may not be dried.
Part B: Liquid bromine (about 41.6 g) is added slowly to a slurry of 2-amino- 6-phenyl-4(3H)-pyrimidine (about 45.0 g on a dry basis) in acetic acid (273.7 ml) and water (273.0 ml) until the reaction is judged to be complete by HPLC. The reaction is quenched with the addition of 50% sodium hydroxide (36.2 g). The reaction mixture is heated to 75-90°C for 1 or more hours. The slurry is cooled and filtered. The cake is washed with water and dried to yield the nonhydroscopic crystal form of bropirimine.
The mode of administration of the compounds of Formula I can be by the means disclosed in US Patent 5,434,157, preferably orally. The dosage administered will be dependent upon the condition of the patient, its age, health, weight, kind of concurrent treatment, if any, frequency of treatment, therapeutic ratio, and tolerance. In addition, the timing of drug administration can be important in maintenance of activity in the treatment of multiple sclerosis.
Illustratively, dosage levels of the administered compounds of Formula I (i.e. "active ingredients") can be intravenous, 0.1 to about 50 mg/kg.; subcutaneous, 0.1 to about 150 mg./kg.; intramuscular, 0.1 to about 150 mg/kg.; and about 0.1 to about 400 mg/kg orally, preferably 15 to about 150 mg/kg of animal (body) weight.
The active ingredients to be employed in the treatment of MS can be easily prepared in unit dosage form with the employment of pharmaceutical materials as disclosed in US Patent 5,434,157.
Illustratively, the active ingredients (compounds of Formula I) are
administered, in divided doses, of about 50 to about 1,000 mg kg/week of human body weight.
A preferred oral dosage regimen of the present invention, is the administration of three grams per day (divided over a six hour period) for three consecutive days per week, e.g. one gram of the active ingredient (preferably presented in unit dosage form) every two hours for three doses on days 1, 2 and 3 of each week for about three to about twelve months, or longer. An alternative preferred oral dosage regimen is the administration of three grams of the active ingredient (preferably presented in unit dosage form) per day (divided over a six hour period) every other day for up to about one year, or longer.
Bropirimine, which is representative of the compounds of Formula I, has been evaluated in an EAE model (Procedure A) and representative results shown in Figures 1-3.
Figure 1: Effect of ABPP on EAE in mice. Mice were dosed with 250 milligrams per kilogram (mpk) on the days indicated by arrows. Two priming doses were administered on consecutive days, and only these doses were given intraperitoneally (i.p.) into mice. Subsequent doses, all were administered orally via a gavage tube followed at 3 or 4 day intervals to result in a twice-a-week dosing regimen. The mean scores for 40 mice in each group were statistically significantly less in ABPP treated mice beginning at day 11 and continued through day 29, that is 7 days after dosing was stopped during the first dosing interval. No significant decline in disease scores was detected during the second dosing interval.
Figure 2: Effect of two different doses of ABPP on EAE in mice. All doses of ABPP were administered orally using a gavage tube. Two priming doses on days 7 and 8 were followed with doses given twice a week for a total of three weeks. The mean disease in mice treated with 400 milligrams per kilogram (mpk) ABPP was statistically significantly less than in mice treated with vehicle alone or with 100 mpk. The mean disease was statistically highly significantly lower for the group treated with 400 mpk beginning on approximately day 15 and continuing through day 37, i.e. approximately 1 week after dosing was stopped. The mean disease for mice treated with 100 mpk ABPP was statistically significantly less than in the vehicle-treated group only on days 16 and 17. Each group began with 65 mice per group, and 5 mice were removed at three times during the course of the experiment for histological evaluation of the lower spinal cords. Additionally, 5 mice from each group were processed for histology on days 43 and 56.
Figure 3: Dose response for ABPP. Two priming doses of ABPP were given
via gavage intubation on days 6 and 7 after injection of MBP to initiate EAE. Dosing was continued at 3 day intervals through day 25. The days that doses were administered to the mice are indicated by arrows just above the abscissa on the graph. Approximately equivalent and statistically significant suppression of disease was observed beginning on day 6 for all 3 doses of ABPP used. After day 16, the degree of disease suppression was greatest in mice treated with 400 mpk ABPP, and the mice treated with 400 mpk had significantly less disease than the mice treated with either 100 mpk or 200 mpk. However, it was difficult to distinguish between the degree of effect of the lower two doses. Disease was suppressed to a statistically significant amount relative to vehicle-treated mice for a period extending for approximately 6 days after the last dose for the 100 mpk and 200 mpk doses and for a period of up to 9 days in mice treated with 400 mpk ABPP.
In the four independent assays, treatment with bropirimine (ABPP) orally, once daily every 3-4 days over 2-3 week periods resulted in a highly statistically significant moderation of EAE. Treatment with bropirimine was begun either 5, 6, or 7 days after induction of disease with myelin basic protein (MBP). Histopathology of formalin-fixed cross sections of the lower spinal cord of mice from the tenth thoracic vertebra to the fourth lumbar vertebra, was also assessed; first, to establish and quantify the correlation between the pattern of the clinical scores and the infiltration of leukocytes and, second, to assess how bropirimine affected the leukocyte infiltration. The results show that there is a small but statistically significant increase in the amount of leukocyte infiltration in the spinal cords of mice with EAE when they are treated with high doses of bropirimine compared to the amount of infiltration expected for mice with equivalent levels of clinical disease but not receiving bropirimine. Procedure A:
Disease Induction 12-20 week old female B10.PL/J mice (Jackson Laboratories, Bar Harbor, Maine) are injected intramuscularly in a rear flank on day 0 with 150 μg of purified guinea pig myelin basic protein (MBP) in complete Freund's adjuvant supplemented with 2.5 mg/ml M. tuberculosis. One day later, 0.1 ml of adsorbed B. pertussis vaccine (Michigan Dept. of Health) is injected subcutaneously at the base of the tail. A second dose of B. pertussis is injected two days later, on day 3. The first signs of disease are usually observed in a few mice beginning at day 6-7 and the mean day of disease onset is approximately day 10. By day 15-20 clinical signs of EAE has been observed in all of the mice.
After induction of EAE with MBP and injection of B. pertussis, the mice are scored approximately 6 times per week for changes in motor activity according to the following scale:
Score 0 = Normal/asymptomatic
1 = limpness or weakness in tail
2 = weakness in one hind limb (hemiparesis/paraparesis)
3 = weakness in both hind limbs (paraparesis)
4 = paralysis in both hind limbs (paraparalysis) 5 = paralysis in both hind limbs, weakness in one or both forelimbs
(paraparalysis/paraparesis) 6 = Death
Dosing for the test compound is via a gavage tube inserted into the stomach of a mouse, unless indicated otherwise. The volume administered is 0.5 ml. The vehicle used with bropirimine (ABPP) contained 5.26 mg carboxymethylcellulose, 4.0 mg polysorbate 80 NF, 9.0 mg sodium chloride, and 9.18 mg benzylalcohol NF.
Histology and Histological Evaluation The mice are perfused with formalin to fix tissues in situ. Eight vertebrae, thoracic 10-13 and lumbar 1-4, are removed from the carcass, further fixed in formalin, decalcified using a 2 x 4 hr incubation in formic acid, carefully separated into individual vertebra and placed in cassettes for paraffin embedding, sectioning, and staining with hematoxylin and eosin (H&E). Five, 5 μm thick spinal cord cross sections are cut, each 100 microns apart, from each of the 8 vertebrae for a total of 40 cross sections per mouse. The cross sections are scored for the presence and degree of infiltration. For an individual mouse, the results are presented as the proportion of cross sections with leukocyte infiltration, regardless of the degree of infiltration, or as the sum of the histological scores for all 40 cross sections. Histology samples were scored according to the following scale: Score
0 = normal histology
1 = 0-10 infiltrating leukocytes in any 1 area
2 = 10-20 infiltrating leukocytes in any 1 area 3 = 20-50 infiltrating leukocytes in any 1 area
4 = 50 or more infiltrating leukocytes in any 1 area
Interferon Assay
Murine interferon determinations are performed using a modification of the method published previously [Stringfellow, D.A., Vanderberg, H.C, Weed, S.D., Interferon induction by 5-halo-6-phenyl pyrimidinones. J. Interferon Res. 1:1-14 (1980)]. L929 (ATCC) cells are used at 90% confluence in 24 well plates. Samples for interferon determination are diluted into DMEM (Gibco) tissue culture medium containing 10% fetal bovine sera and antibiotics. Dilutions of samples are then added to wells of L929 cells and the cells became 100% confluent overnight. The growth medium is removed and the cells are infected with approximately 50 plaque forming units per well of vesicular stomatitis virus (VSV). Virus is allowed to adsorb to cells for one hour, and the wells are overlaid with 0.9% agarose prepared in tissue culture medium containing 2% sera. Cell cultures are incubated for 40-44 hrs, and VSV plaques are counted after 3 hrs of staining with neutral red. Included in each interferon assay are samples of interferon standards obtained from the NIAID Repository, NIH. The standards included murine alpha interferon
(Ga02901511) and a mixture of murine alpha/beta interferon (Gu02901511). In order to quantify the concentration of interferon in samples, inhibition curves generated from the sample dilution series are compared to inhibition curves generated using the interferon standards. Data is reported as units/ml of murine interferon.
Figure 4. Interferon concentrations in peripheral blood in vehicle- and ABPP treated mice. Blood samples were collected on the days indicated and assessed for interferon concentration. Results shown correspond to the experiment shown in Figure 2. Figure 5. Interferon concentrations in peripheral blood in vehicle- and ABPP treated mice. Blood samples were collected on the days indicated and assessed for interferon concentration. Results shown correspond to the experiment shown in Figure 3.
Claims
1. A method for treating multiple sclerosis comprising the systemic administration of an amount effective for treating multiple sclerosis of a compound of the formula:
HO
wherein X3 is equal to X, X4 or X5 wherein X4 is fluoro, chloro, bromo or iodo, and X5 is mono-, di or trihalomethyl, mono, di or trifluoroethyl, perfluoropropyl, and wherein X is alkyl of from 1 to 3 carbon atoms, inclusive, including isomeric forms, 2 propynyl and 2-propenyl, and Xx is a member selected from the group consisting of
(a) phenyl,
(b) a monosubstituted phenyl of the formula
Re
/
-N J
\
Rβ
wherein R5 and R6 are the same or different and are alkyl of from 1 to 8 carbon atoms, inclusive, including isomeric forms, benzyl, or taken together with -N> are a saturated cycloalkylamino group
wherein n' is 3, 4, 5 or 6 or dialkyl substituted cycloalkylamino wherein each alkyl is from 1 to 3 carbon atoms, inclusive; including isomeric forms, and R2 is chloro, fluoro, bromo, iodo, or alkyl of from 1 to 3 carbon atoms, inclusive;
(c) a disubstituted phenyl of the formula:
wherein any two of R, R1; R2, R3 and R4 are not hydrogen and are the same or different and are fluoro, chloro, bromo, iodo, alkyl of from 1 to 8 carbon atoms, inclusive, including isomeric forms, alkoxy of from 1 to 8 carbon atoms, inclusive, including isomeric forms, nitro and trifluoromethyl;
(d) a trihalo substituted phenyl wherein halo is chloro, bromo, iodo, or fluoro;
(e) α-naphthyl of the formula:
wherein R7 is substituted in either ring and is hydrogen, alkyl of from 1 to 8 carbon atoms, inclusive, including isomeric forms, alkoxy of from 1 to 8 carbon atoms, inclusive, including isomeric forms, fluoro, chloro, iodo, bromo or nitro- provided that when X3 is X, Xx is not α-napthyl; (f) 2-furyl, (g) 3-pyridyl, (h) 2-pyridyl, and
(i) 2-pyrazyl, provided that when X3 is X, 2 propynyl or 2-propenyl, Xt is not 2-furyl, 3-pyridyl, 2-pyridyl or 2-pyrazyl, or a salt thereof, in association with a pharmaceutical carrier to a human having symptoms of multiple sclerosis.
2. The method of Claim 1 wherein the amount of compound administered, in divided doses, is from about 50 to about 1,000 mg/kg week. of body weight of the human.
3. The method of Claim 1 wherein the compound administered is selected from the group consisting of:
2-amino-5-iodo-6-(3-bromophenyl)-4-pyrimidinol; 2-amino-5-bromo-6-(3-fluorophenyl)-4-pyrimidinol; 2-amino-5-bromo-6-(3-ethoxyethylphenyl)-4-pyrimidinol;
2-amino-5-bromo-6-(2-methoxyphenyl)-4-pyrimidinol;
2-amino-5-chloro-6-(2-methoxyphenyl)-4-pyrimidinol;
2-amino-5-iodo-6-(2-methoxyphenyl)-4-pyrimidinol;
2-amino-5-bromo-6-(3-chlorophenyl)-4-pyrimidinol; 2-amino-5-iodo-6-(3-chlorophenyl)-4-pyrimidinol;
2-amino-5-chloro-6-(3-chlorophenyl)-4-pvrimidinol;
2-amino-5-chloro-6-(2-fluorophenyl)-4-pyrimidinol;
2-amino-5-chloro-6-(3-fluorophenyl)-4-pyrimidinol;
2-amino-5-bromo-6-(2-fluorophenyl)-4-pyrimidinol; 2-amino-5-iodo-6-(3-fluorophenyl)-4-pyrimidinol;
2-amino-5-iodo-6-phenyl-4-pyrimidinol;
2-amino-5-chloro-6-phenyl-4-pyrimidinol;
2-amino-5-bromo-6-phenyl-4-pyrimidinol;
2-amino-5-chloro-6-(3-methoxyphenyl)-4-pyrimidinol; 2-amino-5-bromo-6-(3-methoxyphenyl)-4-pyrimidinol;
2-amino-5-iodo-6-(3-methoxyphenyl)-4-pyrimidinol;
2-amino-5-bromo-6-(2-pyridyl)-4-pyrimidinol;
2-amino-5-iodo-6-(3,4-dichlorophenyl)-4-pyrimidinol;
2-amino-5-bromo-6-(α-naphthyl)-4-pyrimidinol; 2-amino-5-chloro-6-(3-nitrophenyl)-4-pyrimidinol;
2-amino-5-iodo-6-(3-nitrophenyl)-4-pyrimidinol; 2-amino-5-iodo-6-(3-trifluoromethylphenyl-4-pyrimidinol; 2-amino-5-ethyl-6-phenyl-4-pyrimidinol; 2-amino-5-bromo-6-(3,5-dimethoxyphenyl)-4-pyrimidinol and 2-amino-5-chloro-6-(3-propyloxyphenyl)-4-pyrimidinol.
4. The method of Claim 2 wherein the compound is 2-amino-5-bromo-6-phenyl- 4( lH)-pyrimidinone.
5. The method of Claim 4 wherein the compound is administered orally.
6. The method of Claim 4 wherein the compound is nonhygroscopic.
7. The method of Claim 6 wherein the compound is administered orally.
8. Use of a compound of the formula:
HO
wherein X3 is equal to X, X4 or X5 wherein X4 is fluoro, chloro, bromo or iodo, and X5 is mono-, di or trihalomethyl, mono, di or trifluoroethyl, perfluoropropyl, and wherein X is alkyl of from 1 to 3 carbon atoms, inclusive, including isomeric forms, 2 propynyl and 2-propenyl, and Xx is a member selected from the group consisting of
(a) phenyl,
(b) a monosubstituted phenyl of the formula
R5 /
wherein R5 and R6 are the same or different and are alkyl of from 1 to 8 carbon atoms, inclusive, including isomeric forms, benzyl, or taken together with -N> are a saturated cycloalkylamino group
wherein n' is 3, 4, 5 or 6 or dialkyl substituted cycloalkylamino wherein each alkyl is from 1 to 3 carbon atoms, inclusive; including isomeric forms, and R2 is chloro, fluoro, bromo, iodo, or alkyl of from 1 to 3 carbon atoms, inclusive;
(c) a disubstituted phenyl of the formula:
wherein any two of R, R R2, R3 and R4 are not hydrogen and are the same or different and are fluoro, chloro, bromo, iodo, alkyl of from 1 to 8 carbon atoms, inclusive, including isomeric forms, alkoxy of from 1 to 8 carbon atoms, inclusive, including isomeric forms, nitro and trifluoromethyl;
(d) a trihalo substituted phenyl wherein halo is chloro, bromo, iodo, or fluoro;
(e) α-naphthyl of the formula: 
wherein R7 is substituted in either ring and is hydrogen, alkyl of from 1 to 8 carbon atoms, inclusive, including isomeric forms, alkoxy of from 1 to 8 carbon atoms, inclusive, including isomeric forms, fluoro, chloro, iodo, bromo or nitro- provided that when X3 is X, X1 is not α-napthyl; (f) 2-furyl,
(g) 3-pyridyl,
(h) 2-pyridyl, and
(i) 2-pyrazyl, provided that when X3 is X, 2 propynyl or 2-propenyl, Xx is not 2-furyl, 3-pyridyl, 2-pyridyl or 2-pyrazyl, or a salt thereof, to prepare a medicament to treat multiple sclerosis.
9. The use of Claim 8 wherein the amount of compound administered, in divided doses, is from about 50 to about 1,000 mg/kg/week. of body weight of the human.
10. The use of Claim 9 wherein the compound administered is selected from the group consisting of:
2-amino-5-iodo-6-(3-bromophenyl)-4-pyrimidinol;
2-amino-5-bromo-6-(3-fluorophenyl)-4-pyrimidinol;
2-amino-5-bromo-6-(3-ethoxyethylphenyl)-4-pyrimidinol; 2-amino-5-bromo-6-(2-methoxyphenyl)-4-pyrimidinol;
2-amino-5-chloro-6-(2-methoxyphenyl)-4-pyrimidinol;
2-amino-5-iodo-6-(2-methoxyphenyl)-4-pyrimidinol;
2-amino-5-bromo-6-(3-chlorophenyl)-4-pyrimidinol;
2-amino-5-iodo-6-(3-chlorophenyl)-4-pyrimidinol; 2-amino-5-chloro-6-(3-chlorophenyl)-4-pyrimidinol;
2-amino-5-chloro-6-(2-fluorophenyl)-4-pyrimidinol;
2-amino-5-chloro-6-(3-fluorophenyl)-4-pyrimidinol;
2-amino-5-bromo-6-(2-fluorophenyl)-4-pyrimidinol;
2-amino-5-iodo-6-(3-fluorophenyl)-4-pyrimidinol; 2-amino-5-iodo-6-phenyl-4-pyrimidinol;
2-amino-5-chloro-6-phenyl-4-pyrimidinol; 2-amino-5-bromo-6-phenyl-4-pyrimidinol;
2-amino-5-chloro-6-(3-methoxyphenyl)-4-pyrimidinol;
2-amino-5-bromo-6-(3-methoxyphenyl)-4-pyrimidinol;
2-amino-5-iodo-6-(3-methoxyphenyl)-4-pyrimidinol;
2-amino-5-bromo-6-(2-pyridyl)-4-pyrimidinol;
2-amino-5-iodo-6-(3,4-dichlorophenyl)-4-pyrimidinol;
2-amino-5-bromo-6-(α-naphthyl)-4-pyrimidinol;
2-amino-5-chloro-6-(3-nitrophenyl)-4-pyrimidinol;
2-amino-5-iodo-6-(3-nitrophenyl)-4-pyrimidinol;
2-amino-5-iodo-6-(3-trifluoromethylphenyl-4-pyrimidinol;
2-amino-5-ethyl-6-phenyl-4-pyrimidinol;
2-amino-5-bromo-6-(3,5-dimethoxyphenyl)-4-pyrimidinol and
2-amino-5-chloro-6-(3-propyloxyphenyl)-4-pyrimidinol.
11. The use of Claim 9 wherein the compound is 2-amino-5-bromo-6-phenyl- 4(lH)-pyrimidinone.
12. The use of Claim 11 wherein the compound is administered orally.
13. The use of Claim 11 wherein the compound is nonhygroscopic.
14. The use of Claim 13 wherein the compound is administered orally.
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP52670098A JP2001505911A (en) | 1996-12-12 | 1997-12-03 | How to treat multiple sclerosis |
| CA002269681A CA2269681A1 (en) | 1996-12-12 | 1997-12-03 | Use of a 6-aryl pyrimidine compound for treating multiple sclerosis |
| EP97953042A EP0948331A2 (en) | 1996-12-12 | 1997-12-03 | Method for treating multiple sclerosis |
| AU56871/98A AU5687198A (en) | 1996-12-12 | 1997-12-03 | Method for treating multiple sclerosis |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US3264896P | 1996-12-12 | 1996-12-12 | |
| US60/032,648 | 1996-12-12 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| WO1998025596A2 true WO1998025596A2 (en) | 1998-06-18 |
| WO1998025596A3 WO1998025596A3 (en) | 1998-08-13 |
Family
ID=21866063
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/US1997/021402 WO1998025596A2 (en) | 1996-12-12 | 1997-12-03 | Use of a 6-aryl pyrimidine compound for treating multiple sclerosis |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0948331A2 (en) |
| JP (1) | JP2001505911A (en) |
| AU (1) | AU5687198A (en) |
| CA (1) | CA2269681A1 (en) |
| WO (1) | WO1998025596A2 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000043373A2 (en) * | 1999-01-22 | 2000-07-27 | Amgen Inc. | Kinase inhibitors |
| WO2001068613A1 (en) * | 2000-03-17 | 2001-09-20 | Nissan Chemical Industries, Ltd. | Pyrimidinone derivatives and herbicides |
| US6495558B1 (en) | 1999-01-22 | 2002-12-17 | Amgen Inc. | Kinase inhibitors |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2048250A (en) * | 1979-03-19 | 1980-12-10 | Upjohn Co | 2-amino-6-aryl-4-pyrimidinols and their pharmaceutical uses |
| WO1995011235A1 (en) * | 1993-10-20 | 1995-04-27 | The Upjohn Company | Pyrimidinones as antiarthritic and anti-inflammatories |
| US5434157A (en) * | 1979-03-19 | 1995-07-18 | The Upjohn Company | 6-aryl pyrimidine compounds and method for treating viral infections and inducing interferon production |
-
1997
- 1997-12-03 JP JP52670098A patent/JP2001505911A/en active Pending
- 1997-12-03 CA CA002269681A patent/CA2269681A1/en not_active Abandoned
- 1997-12-03 EP EP97953042A patent/EP0948331A2/en not_active Withdrawn
- 1997-12-03 AU AU56871/98A patent/AU5687198A/en not_active Abandoned
- 1997-12-03 WO PCT/US1997/021402 patent/WO1998025596A2/en not_active Application Discontinuation
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2048250A (en) * | 1979-03-19 | 1980-12-10 | Upjohn Co | 2-amino-6-aryl-4-pyrimidinols and their pharmaceutical uses |
| US5434157A (en) * | 1979-03-19 | 1995-07-18 | The Upjohn Company | 6-aryl pyrimidine compounds and method for treating viral infections and inducing interferon production |
| WO1995011235A1 (en) * | 1993-10-20 | 1995-04-27 | The Upjohn Company | Pyrimidinones as antiarthritic and anti-inflammatories |
Non-Patent Citations (3)
| Title |
|---|
| D.A. STRINGFELLOW: "6-ARYL PYRIMIDINOLES: INTERFERON INDUCERS-IMMUNOMODULATORS-ANTIVIRAL AND ANTINEOPLASTIC AGENTS" PROGRESS IN CANCER RESEARCH AND THERAPY, vol. 16, 1981, pages 215-228, XP002065068 cited in the application * |
| J.E.F. REYNOLDS: "MARTINDALE THE EXTRA PHARMACOPOEIA" 1996 , ROYAL PHARMACEUTICAL SOCIETY , LONDON XP002065070 "INTERFERONS" see page 653 - page 658 * |
| M. YU ET AL.: "INTERFERON-BETA INHIBITS PROGRESSION OF RELAPSING-REMITTING EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS" JOURNAL OF NEUROIMMUNOLOGY, vol. 64, 1996, pages 91-100, XP002065069 cited in the application * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000043373A2 (en) * | 1999-01-22 | 2000-07-27 | Amgen Inc. | Kinase inhibitors |
| WO2000043373A3 (en) * | 1999-01-22 | 2000-12-28 | Kinetix Pharmaceuticals Inc | Kinase inhibitors |
| US6495558B1 (en) | 1999-01-22 | 2002-12-17 | Amgen Inc. | Kinase inhibitors |
| WO2001068613A1 (en) * | 2000-03-17 | 2001-09-20 | Nissan Chemical Industries, Ltd. | Pyrimidinone derivatives and herbicides |
Also Published As
| Publication number | Publication date |
|---|---|
| EP0948331A2 (en) | 1999-10-13 |
| JP2001505911A (en) | 2001-05-08 |
| CA2269681A1 (en) | 1998-06-18 |
| AU5687198A (en) | 1998-07-03 |
| WO1998025596A3 (en) | 1998-08-13 |
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