Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/19—Cytokines; Lymphokines; Interferons
  • A61K38/20—Interleukins [IL]
  • A61K38/208—IL-12
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• This invention is concerned with a novel pharmaceutical composition. More particularly, this invention is concerned with a synergistic antitumor pharmaceutical composition comprising an effective amount of interleukin- 12 (IL-12) and a pyrimidine nucleoside, as well as a hydrate or solvate thereof, that is converted into fluorouracil or its derivative, and pharma ⁇ ceutically acceptable carrier, a synergistic antitumor pharmaceutical composition for the treatment of various cancer and a method of treating various cancers.
• IL-12 interleukin- 12
• a pyrimidine nucleoside as well as a hydrate or solvate thereof, that is converted into fluorouracil or its derivative
• pharma ⁇ ceutically acceptable carrier a synergistic antitumor pharmaceutical composition for the treatment of various cancer and a method of treating various cancers.
• doxifluridine 5'-Deoxy-5-fluorouridine
• doxifluridine a pyrimidine nucleoside
• Doxifluridine is converted into the active drug 5-FU by pyrimidine nucleoside phosphorylases (PyNPase) in vivo, both thymidine and uridine phosphorylases. Therefore, PyNPase is essential for the efficacy of doxifluridine. In fact, tumors with very low levels of this enzyme were refractory to doxifluridine, and PyNPase gene transfection made the tumors more susceptible to this drug.
• IL-12 up-regulates PyNPase activity in tumor tissues and consequently enhances the antitumor activity of doxifluridine.
• IL-12 also enhanced the activity of 5'-deoxy-5-fluoro-N4-(n- pentylcarbonyl)cytidine (capecitabine), which generates doxifluridine and is then converted to 5-FU by PyNPase.
• IL-12 enhanced the anti ⁇ tumor activity of 5-FU to a lesser extent than the anti-tumor activity of doxifluridine.
• cytokines such as IL-la, TNF-a and IFN-g up-regulate PyNPase activity in tumor cell cultures and consequently enhance the susceptibility to doxifluridine (cf. Eda et al. Cancer Chemother Pharmacol. (1993) 32:333-339, and Jpn. J. Cancer Res. 84, 341- 347, March 1993).
• cytokines when given parenterally are distributed to various normal tissues through the circulation and cause systemic side effects, such as flu-like syndrome, leukopenia, hypotension, etc.
• these cytokines distributed to normal tissues as well as tumor tissues would enhance PyNPase activity there and make both the normal and tumor tissues more susceptible to doxifluridine. Therefore, these cytokines would enhance both the efficacy and toxicity of doxifluridine when given in combination.
• IL-12 given parenterally however, induced much higher levels of IFN-g in tumor tissues than in normal tissues. Therefore, IL-12 given parenterally enhances PyNPase activity preferentially in tumor tissues without causing IFN-g-associated systemic side effects.
• the pyrimidine nucleoside is an uridine, cytidine or its derivative represented by the following formula (I) or (II), respectively
• R ⁇ is hydrogen or an radical which is easily hydrolyzable under physiological conditions
• R-2 is hydrogen, cyano, fluorine,lower alkyl or lower alkylidene which may be substituted with one or two fluorine atom(s), or O l
• B ⁇ is lower alkyl, h droxymethyl, or CH2OR 1 , as well as a hydrate or solvate thereof.
• Preferred radicals which are easily hydrolyzable under physiological conditions of Rl in the above formulae (I) and (II) are R ⁇ CO-, R ⁇ OCO- or R SCO- wherein R 4 is alkyl, cycloalkyl, aralkyl or aryl.
• preferred alkyl, cycloalkyl, aralkyl or aryl radical represented by R 4 are a saturated, straight or branched hydrocarbon radical [wherein the number of carbon atoms in the longest straight chain of this hydrocarbon radical ranges from three to seven], or a radical of the formula (CH2)n-Y[ n which n is an integer from 0 to 4, when Y is cyclohexyl, or n is an integer from 2 to 4, when Y is lower alkoxy having 1 to 4 carbon atom(s) or phenyl] .
• a saturated, straight or branched hydrocarbon radical [wherein the number of carbon atoms in the longest straight chain of this hydrocarbon radical ranges from three to seven]" preferably signifies n-propyl, l-isopropyl-2-methylpropyl, 1,1,2-trimethylpropyl, n-butyl, isobutyl, 2-ethylbutyl, 3,3-dimethylbutyl, n-pentyl, isopentyl, neopentyl, 2-propylpentyl, n-hexyl, 2-ethylhexyl, n-heptyl, allyl, 2-buten-l-yl, 3-buten-l-yl, 3-penten-l-yl, 4-penten-l-yl, 3-hexen-l-yl, 4-hexen-l-yl, 5 hexen-1-yl, and the like.
• a radical of the formula (CE_2)n-Y [in which n is an integer from 0 to 4, when Y is a cyclohexyl radical, or n is an integer from 2 to 4, when Y is a lower alkoxy radical having from 1 to 4 carbon atom(s) or a phenyl radical]" preferably siginifies cyclohexyl, cyclohexylmethyl,
• Preferred pyrimidine nucleoside for the present invention are:
• IL-12 is a heterodimeric cytokine which is produced by antigen presenting cells and serves as a pivotal regulator of T and NK cell function (cf. Stern, A.S. et al. Proc. Natl. Acad. Sci. USA. (1990) 87, 6808-6812 and Kobayashi, M. et al. J. Exp. Med. (1989) 170, 827-845).
• Biological activities associated with IL-12 include its ability to enhance the lytic activity of natural killer/lymphokine activated killer cells, to induce the secretion of interferon-g (IFN-g) by both resting and activated T and NK cells, to stimulate the proliferation of activated T and NK cells, to facilitate cytotoxic T lymphocyte responses and to play a critical and unique role in promoting Th-1 type cytokine responses, thereby facilitating cell-mediated immunity (cf. Brunda, M. J. J. Leukocyte Biol. (1994) 55, 280-288 and Taniguchi, G. Blood (1994) 84, 4008-4027).
• IFN-g interferon-g
• IL-12 both human type and murine type, is composed of two disulfide- bonded glycoprotein subunits approximately 35 KDa and 40 KDa in size.
• cDNAs encoding each subunit of IL-12 have been cloned and coexpressed in Chinese Hamster Ovary (CHO) cells to yield the secreted, bioactive, heterodimeric lymphokine (Gubler, U. et al. Proc. Natl. Acad. Sci. USA. (1991) 88, 4143-4147 and Schaenhaut, D.S. et al. J. Immunol. (1992) 148, 3433- 3440)
• a clone of transfected CHO cells secreting recombinant IL-12 was selected.
• Recombinant IL-12 was purified from the culture supernatant of CHO cells grown in a serum-free medium, by ion exchange and gel filtration chromatography.
• the pharmaceutical compostion of the present invention can be administered in any form, for example, tablets, pills, suppositories, capsules, granules, powders, etc. or emulsions.
• the pharmaceutical composition of the present invention are especially suitable for intramuscular, subcutaneous, or intravenous administration.
• Pharmaceutically acceptable carriers and excipients useful in formulating the pharmaceutical composition of the present invention are those commonly used.
• Pharmaceutically acceptable materials can be an organic or inorganic inert carrier material suitable for enteral, percutaneous or parenteral administration such as water, gelatine, gum arabic, lactose, starch, magnesium stearate, talc, vegetable oils, polyalkylene glycols and petroleum jelly.
• the pharmaceutical composition provided by the present invention can be administered orally, e.g.
• the administration can also be carried out parenterally, e.g. in form of sterile solutions, suspensions or emulsions; or locally, e.g. in form of solutions, suspensions, salves, powders or aerosols.
• the pharmaceutical composition can be sterilized and/or can contain further adjuvants such as preserving, stabilizing, setting, emulsifying agents, flavor-improving, salts for variation of the osmotic pressure or substances acting as buffers.
• the synergistic antitumor pharmaceutical composition of the present invention comprises a single pharmaceutical composition as well as a kit of pharmaceutical compositions each containing the individual active ingredient in a desirable dosage form, thus, the present invention is also concerned with a kit for the treatment of colorectal cancer, breast cancer, stomach cancer, lung cancer, cervical cancer, bladder cancer and other malignant diseases which comprises as a first pharmaceutical composition containing an effective amount of IL-12 and a pharmaceutically acceptable carrier, and as a second pharmaceutical composition containing an effective amount of pyrimidine nucleoside derivative, as well as hydrate or solvate thereof and a pharmaceutically acceptable carrier.
• Dosage ranges for the pharmaceutical composition of the present invention can easily be determined by one skilled in the art, and depend on the route of administration, the age, weight and condition of the patient and the particular disease to be treated.
• an approximate range from about 0.05 mg/body/day to about 500 mg/body/day of IL-12, and about 50 mg/body/day to about 20,000 mg/body/day of pyrimidine nucleoside generally range from about 1:100 to about 1:400,000.
• a weight ratio from about 1:1,000 to about 1:10,000 is preferred.
• Rectal administration and intravenous injection are preferred routes of administration of the pharmaceutical composition according to the present invention.
• compositions of the present invention are useful for the treatment of colorectal cancer, breast cancer, stomach cancer, lung cancer, cervicial cancer, bladder cancer, and other malignant diseases and the like.
• mouse IL-12 (mIL-12) at 0.1 mg/mouse or vehicle (0.1 mg/ml of mouse serum albumin dissolved in phosphate-buffered saline) daily for 7 days starting at day 7 and day 8, respectively, after the tumor inoculation.
• mIL-12 mouse IL-12
• vehicle 0.1 mg/ml of mouse serum albumin dissolved in phosphate-buffered saline
• PyNPase activity in the tumor tissues was measured as described in Eda et al. Cancer Chemother. Pharmacol. 1993; 32:333.
• Mouse IFN-g (m IFN-g) levels in the tumor tissue were also measured by a commercially available ELISA system (Intertest g, Genzyme).
• mIL-12 enhanced PyNPase activity 11.9 fold in A75 ⁇ tumors and 2.4 fold in Meth A tumors. This is probably the result of the up- regulation of mIFN-g, which is an up-regulator of PyNPase.
• mice were inoculated with A7 ⁇ mammary adenocarcinoma (2 x 10 5 cells). The mice were given s.c. mIL-12 at 0.1 mg/mouse or vehicle (0.1 mg/ml of mouse serum albumin dissolved in phosphate-buffered saline) daily for 7 days starting at day 6 after the tumor inoculation. One day thereafter, mIFN-g levels in the serum and tissue homogenates of tumor and other organs were measured by ELISA system as mentioned above.
• mIL-12 greatly increased mIFN-g levels in tumors as compared with those in normal organs.
• the tumor tissue level of mIFN-g is 3 to 7 fold higher than those of normal tissues so far examined and 50 fold higher than those in the circulation.
• A7 ⁇ (2 x 10 5 cells) was inoculated s.c. into female C ⁇ 7BL/6 mice.
• the mice were given mIL-12 (0.03 ⁇ g/mouse, s.c), doxifluridine (1.5 mmol/kg, p.o.) and their combination, daily for 4 weeks, starting from day 9 after the tumor inoculation.
• Doxifluridine as a single agent did not show activity in tumor growth inhibition because A75 ⁇ tumor has only low levels of PyNPase, whereas mIL-12 was effective (Table 3). mIL-12 in combination with doxifluridine was much more effective than mIL-12 alone and regressed the tumor.
• Control 28 (23 - 42) 0 o/ ⁇ doxifluridine 29 (22 - 36) 4 0/5
• Meth A fibrosarcoma (5 x 10 5 cells) was inoculated s.c. into male BALB/c mice.
• the mice were given doxifluridine (O. ⁇ mmol/kg, p.o.), capecitabine (1.0 mmol/kg, p.o.), ⁇ -fluorouracil (0.07 ⁇ mmol kg, p.o.), mIL-12 (0.03 ⁇ g/mouse, s.c), and their combination, daily for 3 weeks, starting from day 8 after the tumor inoculation.
• Example 1 illustrate a pharmaceutical preparation of the present invention and do not limit the scope of the present invention.
• a kit having the following components A and B for treatment of colorectal cancer was manufactured in a conventional manner: Component A

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• 1996
  • 1996-05-15 WO PCT/EP1996/002088 patent/WO1996037214A2/en not_active Application Discontinuation
  • 1996-05-15 CN CN96194016A patent/CN1184430A/zh active Pending
  • 1996-05-15 EP EP96917379A patent/EP0828508A2/en not_active Withdrawn
  • 1996-05-15 BR BR9608779A patent/BR9608779A/pt not_active Application Discontinuation
  • 1996-05-15 AU AU59982/96A patent/AU5998296A/en not_active Abandoned
  • 1996-05-15 CA CA002220837A patent/CA2220837A1/en not_active Abandoned
  • 1996-05-15 TR TR97/01435T patent/TR199701435T1/xx unknown
  • 1996-05-15 JP JP8535342A patent/JPH10506641A/ja active Pending
  • 1996-05-22 AR ARP960102652A patent/AR005417A1/es unknown

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