WO1998013047A1 - Utilisations therapeutiques ameliorees de derives d'acide 4-quinoline-carboxylique - Google Patents

Utilisations therapeutiques ameliorees de derives d'acide 4-quinoline-carboxylique Download PDF

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WO1998013047A1
WO1998013047A1 PCT/US1997/017271 US9717271W WO9813047A1 WO 1998013047 A1 WO1998013047 A1 WO 1998013047A1 US 9717271 W US9717271 W US 9717271W WO 9813047 A1 WO9813047 A1 WO 9813047A1
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brequinar
carboxylic acid
quinoline
pyrimidine
uridine
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PCT/US1997/017271
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English (en)
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Anita Chong
Xiulong Xu
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Williams, James, W.
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Priority to AU45960/97A priority Critical patent/AU4596097A/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof

Definitions

  • the present invention relates generally to improved methods of using 4-quinoline-carboxylic acid derivatives, particularly brequinar, for the treatment of various medical conditions, including cancer, inflammatory disorders, and organ transplantation rejection.
  • Cinchophen, 2-phenyl-4-quinoline carboxylic acid has been known for many years and has been described as being useful as an antirheurhatic and in the treatment of gout. However, it can produce a possibly precancerous degeneration of liver tissue.
  • Other 4-quinoline carboxylic acid derivatives have been reported to have anti-neopiastic, anti- inflammatory and immunosuppressive effects, and to be useful for treating cancer, inflammatory disorders, and organ transplant rejection.
  • U.S. Patent No. 4,680,299 (Hesson) reports the use of 2-phenyl-4-quinoline carboxylic acid derivatives for inhibiting the growth of mammalian tumors.
  • Ackerman et al. the combination has a synergistic effect and results in fewer and less severe side effects than does use of immunosuppressive agents alone.
  • U.S. Patent No. 4,861,783 reports the use of substituted quinoline carboxylic acid derivatives in treating skin and muco-epithelial diseases.
  • U.S. Patent No. 4,968,702 (Poletto et al.) addresses substituted quinoline carboxylic acid derivatives, methods for their use in treatment of arthritis and other joint degenerative diseases, and their use in inducing immunosuppression.
  • 5,428,040 reports the use of fused-ring quinoline carboxylic acid derivatives in the treatment of organ transplantation rejection and cancer, either alone or in combination with (1) one or more immunosuppressive agents, or (2) non-steroidal anti- inflammatory agents, or (3) tumor inhibiting agents, i.e, 5-fluorouracil.
  • Brequinar was reported to have activity against L1210 leukemia, P388 leukemia, colon 38 carcinoma and B16 melanoma tumors in vivo in mice as well as have activity against several human cancer cell lines (LX-1 lung, HCT-15 colon, MX-1 breast, BL-STX-1 stomach and CX-1 colon) grafted into animals.
  • DHO-DHase dihydroorotate dehydrogenase
  • Brequinar has been described to exhibit a number of immunosuppressive activities. In vitro, brequinar inhibits T and B cell proliferation, as well as antibody production. In vivo, brequinar suppresses the induction of contact sensitivity, the generation of cytotoxic T cells and the production of antibody. Brequinar also prevents the rejection of allografts and xenografts in a number of rodent transplant models. Because clonal expansion of lymphocytes is central to the induction of both humoral and cellular immune responses, it became widely accepted that immunosuppression by brequinar is mediated through inhibition of pyrimidine synthesis. [Makowka et al., Immunol. Rev.
  • brequinar as an anti-neoplastic, anti- inflammatory or immunosuppressive agent has been limited by its toxic side effects, including bone marrow suppression, which manifests as anemia, leukopenia, granulocytopenia, thrombocytopenia, or bone marrow hypoplasia.
  • bone marrow suppression which manifests as anemia, leukopenia, granulocytopenia, thrombocytopenia, or bone marrow hypoplasia.
  • the maximum tolerated dose was 300 mg/ ⁇ r daily for 5 days.
  • the maximally tolerated doses reported in Schwartsmann et al., Cancer Chemother. Pfiarmacol. , 25:345-351 (1990) were 2,250 mg/m 2 for good risk patients and 1,500 mg/m 2 for poor risk patients, given by a brief intravenous infusion once every 3 weeks.
  • the starting dose of 1800 mg/m 2 given intravenously weekly resulted in unacceptable toxicity, and median weekly doses were reduced to 1200 mg/m 2 .
  • the non-hematological dose-limiting toxicities were largely mucocutaneous side effects such as dermatitis, mucositis, stomatitis and skin rash, which could result in sloughing of skin.
  • Other side effects included nausea, vomiting, fatigue, reversible elevation of liver enzymes and diarrhea.
  • the present invention provides improved methods of using 4- quinoline-carboxylic acid derivatives, particularly brequinar, for the prophylactic or therapeutic treatment of various medical conditions, including cancer, arthritis, inflammatory disorders, and organ transplantation rejection.
  • a pyrimidine such as uridine
  • the co-administration of a pyrimidine, such as uridine, •with the 4-quinoline-carboxylic acid derivative reduces its toxicity while maintaining its therapeutic effectiveness.
  • the brequinar may also be administered in conjunction with other known anti-neoplastic, anti-inflammatory or immunosuppressive agents.
  • a further aspect of the invention involves use of a pyrimidine for the manufacture of a medicament for co-administration with a 4-quinoIine- carboxylic acid derivative, and 4-quinoline-carboxylic acid derivative for the manufacture of a medicament for co-administration with a pyrimidine, as well as use of a pyrimidine and a 4-quinoline-carboxylic acid derivative together for the manufacture of a medicament.
  • Figure 1A depicts hematocrits at 3, 4 and 6 weeks of mice treated with nothing (control) or brequinar at 10 or 20 mg/kg, reported as % packed cell volume (PCV).
  • Figure IB depicts hematocrits of mice (% PCV) at six weeks after treatment with various doses of brequinar or a combination of brequinar and uridine.
  • Figures 2A and 2B depict the average weight of the lymph nodes and spleen, respectively, for mice treated with brequinar, uridine, or a combination of both.
  • Figure 3 depicts hematocrits of mice (% PCV) at 13, 23, 31 and 53 days after treatment with brequinar, uridine, or a combination of both.
  • the present invention provides improved methods of using 4- quinoline-carboxylic acid derivatives for the treatment of various medical conditions, including cancer, arthritis, inflammatory disorders, including auto- immune diseases, and organ transplantation rejection, including graft vs. host disease.
  • the co-administration of a pyrimidine with the 4-quinoline-carboxylic acid derivative reduces its toxicity while maintaining its therapeutic effectiveness.
  • a "4-quinoIine- carboxylic acid derivative" is defined to include those compounds described as having anti-neoplastic, anti-inflammatory and/or immunosuppressive activity in U.S. Patent Nos. 4,680,299 (e.g. , at col. 4, line 30 to col. 8, line 40), 4,847,381 (e.g.
  • a preferred 4-quinoline-carboxylic acid derivative is brequinar.
  • a "pyrimidine” includes compounds useful either directly or as intermediates in pathways for supplying pyrimidine nucleotides (uridine, cytidine and thymidine).
  • a preferred pyrimidine is uridine.
  • Other suitable pyrimidines include the pyrimidine intermediates orotic acid and orotidine.
  • Other exemplary pyrimidines include cytidine and thymidine, possibly at higher doses.
  • the invention is based in part on the discovery that the in vivo pharmacological activity of 4-quinoline-carboxylic acid derivatives is mediated by their inhibition of tyrosine kinase phosphorylation, which plays an important role in the activation and proliferation of lymphocytes following the stimulation of antigen and cytokine receptors, and that the activity of these drugs is largely independent of their inhibition of the DHO-DHase enzyme in the pyrimidine synthetic pathway.
  • This discovery is directly contrary to the prevailing belief that the pharmacological activity of these compounds is mediated by the inhibition of pyrimidine synthesis. In fact, the inhibition of pyrimidine synthesis results in suppression of bone marrow hematopoiesis and is responsible for the toxicity of these compounds. This toxicity can effectively be ameliorated by administration of a pyrimidine, without impairing the therapeutic effectiveness of the 4-quinoline-carboxylic acid derivatives.
  • results described herein demonstrate that, in a mouse allograft model of transplant rejection and in a mouse model of autoimmune disease and lymphoproliferative disorder, immunosuppression by a 4- quinoline-carboxylic acid derivative, brequinar, is largely independent of the effects on pyrimidine synthesis, while brequinar is active as an inhibitor of protein tyrosine phosphorylation.
  • results also show that co-administration of a pyrimidine, uridine, with brequinar effectively eliminates the toxicity associated with the inhibition of pyrimidine synthesis while maintaining brequinar's immunomodulatory activity in the transplant rejection, autoimmune disease and lymphoproliferative disorder models.
  • the improved methods of the present invention are useful for the treatment of subjects undergoing a variety of organ transplants, including kidney, liver, heart, lung, skin, bone marrow and pancreatic islet cells. These improved methods are also useful for the treatment of neoplastic diseases, including leukemia, breast cancer, lung cancers including small cell lung cancer, colon cancers, stomach cancer, melanoma, other skin cancers, and brain cancers.
  • organ transplants including kidney, liver, heart, lung, skin, bone marrow and pancreatic islet cells.
  • neoplastic diseases including leukemia, breast cancer, lung cancers including small cell lung cancer, colon cancers, stomach cancer, melanoma, other skin cancers, and brain cancers.
  • the 4-quinoline-carboxylic acid derivative and the pyrimidine may be administered systemically or topically.
  • Systemic routes of administration include oral, intravenous, intramuscular or subcutaneous injection (including into depots for long-term release), intraocular or retrobulbar, intrathecal, intraperitoneal (e.g. by intraperitoneal injection or lavage), intrapulmonary using aerosolized or nebulized drug, or transdermal using, e.g., patches.
  • Topical routes include administration in the form of salves, creams, jellies, ointments, ophthalmic drops or ointments, ear drops, suppositories, or irrigation fluids (for, e.g., irrigation of wounds).
  • 4-quinoline-carboxylic acid derivative compositions are generally administered in doses ranging from 1 ⁇ g/kg to 500 mg/kg per day, or preferably in doses ranging from 2 mg/kg to 50 mg/kg per day, or in equivalent dosing at longer or shorter intervals.
  • higher dosages of the 4-quinoline-carboxylic acid derivative over a shorter treatment period may be preferred.
  • Brequinar is water soluble and has been reported to have very good oral bioavailability, reaching blood levels comparable to (greater than 90% of) the levels achievable by intravenous injection. [Arteaga et al., Cancer Res. , 49:46-48 (1989); Sherls et al., Hepatology, 18:746 (1993).] Brequinar has previously
  • mice were administered to humans intravenously at doses ranging from 15 mg/m to a maximal tolerated dose of 2,250 mg/m 2 (a 70 kg man has a surface area of approximately 1.73 m 2 ).
  • Prior studies in mice indicated that mice receiving the optimal intravenous dose on a daily schedule for 9 days had a brequinar blood level of 3 g/ml. [Noe et al., supra.] It is contemplated, however, that co-administration with uridine will improve tolerability of brequinar and therefore raise the maximally tolerated dose by at least 5 -fold.
  • Pyrimidine compositions are generally administered in doses ranging from 1 mg/kg to 5000 mg/kg per day, preferably in doses ranging from 50 mg/kg to 200 mg/kg per day given orally, or in equivalent dosing at longer or shorter intervals.
  • Humans with orotic aciduria are generally given uridine supplementation at doses of 150 mg/kg.
  • the doses of the 4-quinoline-carboxylic acid derivative or pyrimidine may be increased or decreased, and the duration of treatment may be shortened or lengthened as determined by the treating physician.
  • the frequency of dosing will depend on the pharmacokinetic parameters of the agents and the route of administration.
  • the optimal pharmaceutical formulation will be determined by one skilled in the art depending upon the route of administration and desired dosage. See for example, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA 18042) pages 1435-1712, the disclosure of which is hereby incorporated by reference. Such formulations may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the administered agents.
  • the specific dose may be calculated according to body weight, body surface area or organ size. Further refinement of the calculations necessary to determine the appropriate dosage for treatment involving each of the above mentioned formulations is routinely made by those of ordinary skill in the art without undue experimentation, especially in light of the dosage information and assays disclosed herein, as well as the pharmacokinetic data observed in the human clinical trials discussed above. Appropriate dosages may be ascertained through use of established assays for determining blood levels dosages in conjunction with appropriate dose-response data.
  • the final dosage regimen will be determined by the attending physician, considering various factors which modify the action of dmgs, e.g. the drug's specific activity, the severity of the damage and the responsiveness of the patient, the age, condition, body weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors. As studies are conducted, further information will emerge regarding the appropriate dosage levels for the treatment of various diseases and conditions.
  • a further aspect of the invention involves use of a pyrimidine for the manufacture of a medicament for co-administration with a 4-quinoIine- carboxylic acid derivative, and 4-quinoline-carboxylic acid derivative for the manufacture of a medicament for co-administration with a pyrimidine, as well as use of a pyrimidine and a 4-quinoline-carboxylic acid derivative together for the manufacture of a medicament.
  • the methods of the present invention can be used with other current standard care, such as with other anti-neoplastic agents for cancer treatment, with other anti-inflammatory or immunosuppressive agents such as corticosteroids or non-steroidal anti-inflammatory drugs (NSAIDs) for treatment of inflammatory disorders, and with antibiotics and other immunomodulatory therapy such as cyclosporine A, FK506, corticosteroids, azathioprine, mycophenolic acid, rapamycin, 15-deoxyspergulin, mizoribine, leflunomide, OKT3, anti-IL-2 receptor antibodies, misoprostol, methotrexate, cyclophosphamide and anti-lymphocyte or anti-thymocyte antibodies, for organ transplantation.
  • other anti-inflammatory or immunosuppressive agents such as corticosteroids or non-steroidal anti-inflammatory drugs (NSAIDs) for treatment of inflammatory disorders
  • antibiotics and other immunomodulatory therapy such as cyclosporine A, FK506, corticosteroids,
  • the methods of the present invention may improve therapeutic effectiveness of these other agents. This may occur through reducing the amount of agent required to obtain the desired clinical effect, thereby lowering systemic toxicity and/or cost of treatment, and thus allowing wider use of the agents.
  • the present invention may also provide quality of life benefits due to, e.g., decreased duration of therapy, reduced stay in intensive care units or reduced stay overall in the hospital, with the concomitant reduced risk of serious nosocomial (hospital-acquired) infections.
  • treating encompasses both prophylactic and therapeutic use.
  • Concurrent administration includes administration of the agents together, or before or after each other.
  • the agents may be administered by different routes.
  • the 4-quinoline-carboxylic acid derivative may be administered intravenously while the pyrimidine is administered intramuscularly, intravenously, subcutaneously or orally.
  • another anti-neoplastic, anti-inflammatory or immunosuppressive agent may also be administered, e.g. , intravenously or orally.
  • the agents may be given sequentially in the same intravenous line, after an intermediate flush, or may be given in different intravenous lines. They may be administered simultaneously or sequentially, as long as they are given in a manner sufficient to allow both agents to achieve effective circulating concentrations.
  • Example 1 addresses the toxic effects of brequinar, with and without uridine, in mice.
  • Example 2 addresses the immunosuppressive effects of brequinar, with and without uridine, in a mouse cardiac allograft transplant model.
  • Example 3 addresses brequinar's effects on tyrosine phosphorylation.
  • Example 4 addresses the immunosuppressive effects of brequinar, with and without uridine, in a mouse lymphoproliferative disorder model.
  • Example 5 addresses the immunosuppressive effects of brequinar, with and without uridine, in a mouse auto-immune disease model.
  • mice Four to six normal Balb/C mice were treated for six weeks with 10 or 20 mg/kg/day brequinar (Dupont Merck Pharmaceuticals, Wilmington, DE) diluted in saline via intraperitoneal injection to induce anemia. Blood and serum samples were obtained from the mice at weeks 3, 4 and 6. Nucleotide triphosphate and uridine levels in the spleen, bone marrow and serum were quantitated as follows. Spleen or bone marrow (60 mg per sample) were briefly homogenized in 540 ⁇ l of 0.4 M tricholoric acid.
  • nucleotides were extracted by centrifugation and then neutralized with equal volume of 0.4 M tri-n-octylamine in Freon 113 as described [Olempska-Beer and Freese, 1984]. Nucleotides were analyzed in Waters HPLC system with a 616 pump, a 600S gradient controller, a 717 plus autosampler and 996 PDA detector (Milford, MA). The separation was achieved by a linear gradient elution of potassium phosphate buffer, pH 4.5 (10 to 550 M) on a Whatman strong anion exchange column, partisil 10 SAX (Alltech, Deerfield, IL).
  • the column was eluted with 5mM KH 2 PO 4 , pH 6.8 containing 5% methanol at a flow of 1 ml/min.
  • Uridine was quantified by measurement of absorbance at 280 nm and 254 nm and comparison of the peak height with that of standard solutions.
  • mice - 13 - hematopoiesis, and induces anemia in Balb/c mice.
  • the hematocrits of these mice reported as % packed cell volume (PCV) in Figure 1A, were significantly reduced to 44-45% after 4 weeks of treatment with 20-10 mg/kg brequinar, compared to 65% in the untreated group. PyNTP levels were also significantly depressed in bone marrow cells after treatment with brequinar.
  • mice treated with 10 or 20 mg/kg/day brequinar were also co-administered 1000 or 2000 mg/kg/day uridine (Sigma, St. Louis, MO) intraperitoneally for six weeks, the uridine prevented anemia, and the hematocrits remained at levels comparable to untreated controls (61-63 %) as shown in Figure IB.
  • the co-administration of uridine returned UTP and CTP levels in bone marrow cells to normal as shown in Table 1 below.
  • Table 1 Effect of uridine co-administration with brequinar on intracellular nucleotides of bone marrow cells in vivo. Nucleotide quantitative data is expressed as ⁇ g nucleotide/mg protein.
  • the immunosuppressive activity of brequinar was monitored in the Balb/c-into-C3H cardiac allograft model. [Corry et al., Transplantation, 16:343 (1973).] Allograft recipients were treated with 2 or 10 mg/kg/day brequinar as monotherapy or in combination with 1000 mg/kg/day uridine, and were monitored for allograft survival. Results are shown below in Table 2. The untreated allografts survived for a mean of 9.2 ⁇ 0.83 days.
  • Co- administration of uridine with either 2 or 10 mg/kg/day brequinar prolonged allograft survival to 51.8 ⁇ 16.5 days or 51.0 ⁇ 16.1 days, respectively, and alleviated the fatal toxicity of the high dose brequinar therapy.
  • T cells isolated from lymph nodes of Balb/c mice were stimulated with 2C11.145 anti-CD3 antibody, 2 ⁇ g per sample, 5 x 10" cells per sample.
  • the cells were pelleted and directly lysed in NP-40 buffer (50 mM Hepes-HCI, pH 8.0; 150 mM NaCl; 1 % Nonidit P-40; 5 mM EDTA; 1 mM sodium vanadate; 5 mM NaF; 1 mM PMSF; 10 ⁇ g of aprotinin and leupetinin each).
  • Protein concentration of the postnuclear lysates was measured by using a Bio-Rad protein assay kit (Bio-Rad Lab., Hercules, CA). Thirty ⁇ g protein of each sample was separated on a SDS-polyacylamide gel, and then transferred onto nitrocellulose membrane. Protein tyrosine phosphorylation was monitored by Western blot using monoclonal antibody 4G10 (UBI, Placid Lake, NY) and enhanced chemiluminescence (ECL).
  • brequinar to inhibit tyrosine kinases in vitro was also tested as follows. Immunoprecipitated p59 fvn or p56 lck from CTLL-4 cells or LSTRA cells (5 x 10 ), respectively, were preincubated with various concentrations of brequinar in the PTK buffer (50 mM Hepes, pH 7.4, 10 mM MgCl 2 , and 10 mM MnCl 2 ) on ice for 10 min. Exogenous substrate histone 2B (2 ⁇ g) was added and after 20 min, the reaction was initiated by addition of 10 ⁇ Ci 32 [P]- ⁇ - ATP.
  • PTK buffer 50 mM Hepes, pH 7.4, 10 mM MgCl 2 , and 10 mM MnCl 2
  • reaction mixture was subject to electrophoresis on a 12.5 % sodium dodecyl sulfate (SDS)-polyacrylamide gel. Autophosphorylation of the kinase and phosphorylation of exogenous substrate were analyzed by autoradiography.
  • SDS sodium dodecyl sulfate
  • Brequinar at a concentration of 25-100 ⁇ M was able to significantly inhibit tyrosine phosphorylation induced in murine T cells by cross-linking with anti-CD3 monoclonal antibodies.
  • the in vitro kinase activities of two src-family kinases, lck ⁇ ndjyn kinases were inhibited with 50-200 ⁇ M brequinar.
  • tissue and serum brequinar concentrations are reported to range from a peak of 200-500 ⁇ M to a 24 hour trough level of 20-50 ⁇ M in mice treated with a single i.v.
  • lymphocytes In MRL-lpr/lpr mice, the lymphocytes have a mutated, inactive Fas transmembrane protein and are constitutively proliferative [Zhou et al., J. Immunol., 150:3651-3667 (1993)]. Although these cells do not have the characteristics of malignancy, their Fas protein mutation results in defective apopotosis, persistent cell accumulation and proliferation mainly localized in thymus and lymph nodes.
  • MRL-lpr/lpr mice were purchased from Jackson Laboratories (Bar Harbor, ME). MRL-lpr/lpr mice, 4 in each group at the age of 10 weeks, were treated with 10 mg/kg/day brequinar alone, 10 mg/kg/day brequinar plus 1000 mg/kg/day uridine, 1000 mg/kg/day uridine alone, for seven weeks by intraperitoneal injection. One group remained untreated, as a control.
  • the 1000 mg/kg/day uridine was given in twice daily doses of 500 mg/kg, since a large dose of uridine would be most likely to prevent brequinar's action if the activity is caused by pyrimidine synthesis inhibition.
  • Four hours after the last treatment with brequinar the mice were sacrificed. Blood samples were collected, and serum samples were prepared and stored at -80°C. Lymph nodes and thymi were also collected. Protein tyrosine phosphorylation was determined as follows.
  • lymph node was directly lysed in NP-40 buffer (50 mM Hepes-HCI, pH 8.0: 150 M NaCl; 1 % Nondit P-40; 5 mM EDTA; 1 mM sodium vanadate; 5 mM NaF; 1 mM PMSF; 10 ⁇ g of aprotinin and leupetinin each) , postnuclear lysates were prepared. Protein concentration in cell lysates was measured by using a Bio-Rad protein assay kit (Bio-Rad Lab, Hercules, CA). Thirty ⁇ g protein of each sample was separated on a SDS- polyacrylamide gel, and then transferred onto nitrocellular membrane.
  • NP-40 buffer 50 mM Hepes-HCI, pH 8.0: 150 M NaCl; 1 % Nondit P-40; 5 mM EDTA; 1 mM sodium vanadate; 5 mM NaF; 1 mM PMSF; 10 ⁇ g of aprot
  • Protein tyrosine phosphorylation was monitored by using western blot and enhanced chemiluminescence (ECL).
  • ECL enhanced chemiluminescence
  • the exposed X-Omat films from the in vitro tyrosine kinase assays or the phosphotyrosine proteins detected on Western blots were scanned in a LKB densitometer (2202 Ultrascan Laser Densitometer). The peaks corresponding to the bands of interest were integrated to determine the relative amounts of phosphorylation.
  • the intracellular nucleotide pool was analyzed as follows.
  • Lymph node or thymus (60 mg per sample) were briefly homogenized in 540 ⁇ l of 0.4 M trichloric acid, nucleotides were extracted by centrifugation and then neutralized with equal volume of 0.4 M tri-n-octylamine in Freon 113. Nucleotides were analyzed in a Waters HPLC system with a 616 pump, a
  • Serum Sample was diluted twofold in 0.9% NaCl, uridine was extracted by addition of equal volume of 0.8 M trichloric acid and then neutralized with equal volume of 0.4 M tri-n- octylamine. The level of serum uridine was quantitated in a Waters HPLC system.
  • the average weight of the lymph nodes and spleen for each treatment group is displayed in Figures 2 A and 2B.
  • Treatment with 10 mg/kg/day brequinar monotherapy for 7 weeks reduced the weight of lymph nodes 10-fold, with a slight decrease in spleen weight as well.
  • Uridine treatment alone had no effect on the weight of either organ.
  • Co-administration of 10 mg/kg/day brequinar with 1000 mg/kg/day uridine significantly reduced the size of lymph nodes by 3-fold.
  • brequinar did not decrease the intracellular pyrimidine nucleotide pools nor the purine nucleotide pools in the lymphocytes prepared from thymus or lymph nodes. This retention of normal levels of pyrimidine nucleotides in the lymphocytes of brequinar-treated MRL-lpr/lpr mice is likely due to the rapid salvage of uridine from serum and/or the activation of uridine kinase that converts intracellular uridine to UTP.
  • This inconsistency may reflect the difference in the dose of brequinar used in the experiments, or a compensatory elevation in DHO-DHase activity after 4 weeks of treatment, or a decreased demand for nucleotides because of reduced proliferation in the lymphoid compartments.
  • the MRL-lpr/lpr mice of Example 4 also experience an auto- immune disease involving auto-antibody deposition which leads to tissue damage and dysfunction in some organs such as kidney, pancreas and salivary gland. Brequinar's therapeutic effect in auto-immune diseases is partly mediated by its inhibition of auto-antibody production.
  • Results of monitoring auto-antibody production in the mice of Example 4 are displayed below in Table 3.
  • the serum samples prepared from sacrificed mice were measured for the content of auto-antibody by using an ELISA assay according to Hou et al., J. Immunol. , 150:3651-3667 (1993).
  • the results show that brequinar can significantly control anti-DNA IgG and IgM production, and that co-administration with uridine had no effects on the inhibition of auto-antibody production in brequinar-treated animals.

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Abstract

Cette invention se rapporte de façon générale à des procédés améliorés d'utilisation de dérivés d'acide 4-quinoline-carboxylique, en particulier brequinar, pour le traitement de divers états pathologiques, tels que le cancer, l'arthrite, les troubles inflammatoires et le rejet de la transplantation d'organes. Ces procédés améliorent l'efficacité thérapeutique et réduisent la toxicité des dérivés d'acide 4-quinoline-carboxylique.
PCT/US1997/017271 1996-09-26 1997-09-26 Utilisations therapeutiques ameliorees de derives d'acide 4-quinoline-carboxylique WO1998013047A1 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6583156B1 (en) * 1998-09-04 2003-06-24 Vernalis Research Limited 4-Quinolinemethanol derivatives as purine receptor antagonists (1)
WO2000033876A1 (fr) * 1998-12-10 2000-06-15 Aventis Pharma Deutschland Gmbh Formulation a spectre therapeutique elargi, contenant des inhibiteurs de la synthese des nucleotides
AU766810B2 (en) * 1998-12-10 2003-10-23 Aventis Pharma Deutschland Gmbh Formulation with an improved therapeutic range, containing nucleotide synthesis inhibitors
WO2017037022A1 (fr) * 2015-09-01 2017-03-09 Bayer Pharma Aktiengesellschaft Composés et méthodes utiles pour le traitement ou la prévention de cancers hématologiques
CN108472280A (zh) * 2015-09-01 2018-08-31 布罗德研究所股份有限公司 用于治疗或者预防血液癌症的化合物和方法
US20180263970A1 (en) * 2015-09-01 2018-09-20 The Broad Institute, Inc. Compounds and methods useful for treating or preventing hematological cancers
US11096934B2 (en) 2015-09-01 2021-08-24 The Broad Institute, Inc. Compounds and methods useful for treating or preventing hematological cancers
US20220016106A1 (en) * 2015-09-01 2022-01-20 The Broad Institute, Inc. Compounds and methods useful for treating or preventing hematological cancers

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