WO1987003489A1

WO1987003489A1 - Combinations of tumor necrosis factors and antibiotics and methods for treating tumors

Info

Publication number: WO1987003489A1
Application number: PCT/EP1986/000710
Authority: WO
Inventors: Walter Charles Fiers; Lucia Marie Fransen; Jose Van Der Heyden
Original assignee: Biogen N.V.
Priority date: 1985-12-05
Filing date: 1986-12-05
Publication date: 1987-06-18
Also published as: JPS63501724A; EP0249618A1

Abstract

Combinations of TNF and antibiotics, such as the broad spectrum antibiotics chloramphenicol or tetracycline, which impede or inhibit protein synthesis, and methods of treatment of tumors by administering pharmaceutically effective amounts of TNF and those antibiotics to a tumor-bearing mammal.

Description

COMBINATIONS OF TUMOR NECROSIS FACTORS AND ANTIBIOTICS AND METHODS FOR TREATING TUMORS

TECHNICAL FIELD OF INVENTION

This invention relates to combinations useful for the enhancement of growth inhibition or killing of tumor cells. More particularly, this invention relates to combinations of natural or recombinant tumor necrosis factors ("TNF") and broad spectrum antibiotics, such as chloramphenicol and tetracycline, which impede or inhibit protein syn¬ thesis in the mitochondria of the target cell. Accord¬ ing to this invention, these antibiotics are used to enhance the growth inhibiting or killing effect of TNFs on tumor cells.

BACKGROUND ART

TNF is produced by macrophages and mono- nuclear phagocytes. It is cytotoxic or cytostatic for a broad range of animal and human cancer cells in vitro and induces hemorrhagic necrosis in certain animal tumors and heterotransplanted human tumors in vivo [K. Haranaka and N. Satomi, "Note: Cytotoxic Activity of Tumor Necrosis Factor (TNF) on Human Cancer Cells in vitro, " Japan J. Exp. Med. , 51, pp. 191-94 (1981); L. Old, "Cancer Immunology: The -2-

Search for Specificity - G. H. A. Clowes Memorial Lecture," Cancer Research, 41, pp. 361-75 (1981)].

Antibiotic agents may be classified on the basis of their chemical structure and mechanism of action. For example, agents that affect the function of ribosomes to inhibit or impede mitochondrial protein synthesis form a class of antibiotics which are useful in the combinations of this invention. This class includes chloramphenicols and tetracy- clines, aminoglycosidic antibiotics, the macrolide antibiotics (i.e., erythromycin), linco ycin, and its congener clindamycin.

Tetracyclines comprise a class of broad- spectrum antibiotics with a wide-range of anti- microbial activity. They are metabolites or semi- synthetic derivatives of metabolites of Streptomyces aureofaciens. They have bacteriostatic, or, at high concentrations, bacteriocidal effects on many species of gram-positive and gram-negative bacteria, spiro- chetes, and rickettsiae. The site of action of tetracyclines is the ribosome. They inhibit protein synthesis by preventing the addition of amino acids to a growing peptide chain. Tetracyclines also impair protein synthesis in mammalian cells at high concen- trations [A. Goodman et al., The Pharmacological

Basis of Therapeutics, pp. 1181-99 (1980) ("Goodman and Gilman's")] .

Chloramphenicol is an antibiotic produced by Streptomyces venezuelae. It is a broad spectrum antibiotic which also inhibits protein synthesis in bacteria by binding to the ribosomes of its target. Chloramphenicol can also inhibit mitochondrial protein synthesis in mammalian cells probably because mito¬ chondrial ribosomes resemble bacterial ribosomes [L. . Wheeldon and A. L. Lehninger, Biochemistry, 5, pp. 3533-45 (1966)]. -3-

The a inoglycoside antibiotics include gentamicin, tobramycin, amikacin, kanamycin, strepto¬ mycin and neomycin. These antibiotics act directly on the ribosome, where they inhibit protein synthesis and decrease the fidelity of translation of the genetic code [Goodman and Gil an's, supra, pp. 1162-80].

Erythromycin and other macrolide anti¬ biotics, lincomycin, and clindamyσin inhibit protein synthesis by binding to 50S ribosomal subunits.

Although they are not structurally related, erythro¬ mycin, chloramphenicol and clindamycin can interfere with each other's binding at this site [Goodman and Gilman's, supra, pp. 1222-27]. Conventional treatment of tumors include non-surgical treatments, such as chemotherapy and radiation, and surgical treatments. Typically, these treatments are characterized by various undesirable side effects. Non-surgical treatments having immuno- suppressant effects may increase the patient's susceptibility to secondary infections. Surgical treatments to excise transformed cells involve risks attendant with invasive procedures and may not effec¬ tively remove or eliminate the entire transformed cell population. Alternative methods of treatment for cancers and non-malignant tumors have involved the use of monoclonal antibodies to tumor specific antigens on the surface of transformed cells. The effectiveness of such treatments, typically involving murine monoclonal antibodies, is often limited by a variety of factors, including anti-antibody responses which impede the effectiveness of further adminis¬ trations of the murine antibody [G. E. Goodman et al., "Pilot Trial of Murine Monoclonal Antibodies In Patients With Advanced Melanoma", Journal Of Clinical Oncology, 3_, pp. 340-51 (1985)]. Other reported side effects of monoclonal antibody treatments include anaphylaxis, fever and chills.

In view of the disadvantages of such thera¬ pies, various therapies have been directed to aug- - — meriting the body's immune response to tumorigenic cells by increasing the body's level of various lymphokines. For example, TNF alone is known to inhibit the growth of or to kill tumor cells. In addition, combinations of human lymphotoxin and human gamma interferon have been reported to inhibit tumor growth [European patent application 128,009]. Combi¬ nations of TNF and human interferon have also been reported to demonstrate a greater growth inhibitory or cytotoxic effect on human tumors than the sum of their separate effects [L. Fransen et al., "Recom- binant Tumor Necrosis Factor: Its Effect And Its Synergism With Interferon-γ On A Variety Of Normal And Transformed Human And Mouse Cell Lines" (in press); see also European Patent Application 131,789].

DISCLOSURE OF THE INVENTION

The present invention provides combinations and methods that cause the inhibition of tumor growth or the enhancement of tumor cell death to a far greater degree than TNF alone. Surprisingly, in accordance with this invention, broad spectrum anti¬ biotics which inhibit or impede protein synthesis in the target cell dramatically enhance the tumoricidal effect of TNF. Particularly useful combinations with such TNF treatments include the tetracyclines or chloramphenicol.

BEST MODE OF CARRYING OUT THE INVENTION

In order that the invention herein described may be more fully understood, the following detailed description is set forth. -5-

In the description the folowing terms are employed:

TNF (or tumor necrosis factor) — TNF is a growth inhibitory or cytotoxic lymphokine. Natural TNF is a protein with a molecular weight of over 17,000. TNF has been produced in small quantities in vivo. For example, endotoxin may be used to trigger the release of TNF by activated macrophages. TNF can also be induced in established cell lines, i.e., U937 [D. J. Camerson, Reticuloenthel. Soc. , 34, pp. 45-52 (1983)]. TNF has been cloned and expressed in various host-vector systems [A. L. Mar enout et al., "Molecular Cloning And Expression Of Human Tumor Necrosis Factor And Com- parison With Mouse Tumor Necrosis Factor, " Eur. J.

Biochem, 152, pp. 515-22 (1985); and L. Fransen et al., "Molecular Cloning Of Mouse Tumour Necrosis Factor cDNA And Its Eukaryotic Expression, " Nucl. Acid Res. , 13, pp. 4417-29 (1985); see also D. Pennica et al., Nature, 312, pp. 724-28 (1984); T. Shirai, Nature, 313, pp. 803-06 (1985); A. M. Wang et al., Science, 228, pp. 149-54 (1985)].

The nucleotide sequence of cloned TNF indi¬ cates that it is composed of approximately 157 amino acids. As used in this application, "TNF" includes all proteins, polypeptides, and peptides which are natural or recombinant TNFs, or derivatives thereof, and which are characterized by the tumoricidal activity of these TNFs. They include TNF-like com- pounds from a variety of sources, such as natural TNFs, recombinant TNFs, and synthetic or semi-syn¬ thetic TNFs.

Tumor — As used in this application, the term "tumor" encompasses any undesirable proliferation of cells. Such proliferation includes malignant and non-malignant, solid or fluid tumors, carcinomas, -6- myelomas, sarcomas, leukemias, lymphomas,. and other cancerous, neoplastic, or tumorigenie diseases.

Chloramphenicol — Chloramphenicol is an antibiotic which impedes protein synthesis in bacteria and mammalian cells by attaching to bacterial ribo¬ somes or mitochondrial ribosomes, respectively, in order to cause cell death. As used in this applica¬ tion, "chloramphenicol" includes related derivatives, such as thioamphenicol, a more water-soluble and less toxic derivative of chloramphenicol.

Tetracycline — Tetracyclines are a class of antibiotics which impede protein synthesis in bacteria and mammalian cells by attaching to bacterial ribosomes or mitochondrial ribosomes, respectively, in order to cause cell death. As used in this appli¬ cation, "tetracycline" includes related members of the broad family of antibiotics generally known as tetracycline, as well as their derivatives. The term includes, for example, chlortetracycline, oxy- tetracycline, demeclocycline, methacycline, doxycycline and minocycline.

This invention relates to combinations and methods for treating tumors and neoplastic diseases. More particularly, this invention relates to combi- nations of pharmaceutically effective amounts of TNF and pharmaceutically effective amounts of broad spectrum antibiotics that inhibit or impede protein synthesis in the target cell. In the preferred embodiment of this invention, those antibiotics are selected from the group of tetracyclines or chloramphenicol.

Among the TNFs useful in the combinations and treatments of this invention are the TNFs produced in vitro by a variety of cells in response to various inducers. For example, these TNFs include compounds displaying TNF activity obtained from sera of mice -7- and rabbits which have been infected with Bacillus- Cal ette-Guerin (BCG) or Corynebacterium and treated with lipopolysaccharide (LPS) of Escherichia coli [E. A. Carswell et al., "An Endotoxin-Induced Serum Factor That Causes Necrosis Of Tumors", Proc. Natl. Acad. Sci. USA, 72, pp. 3666-70 (1975)]. Also useful are the TNFs derived from the incubation media of macrophage-enriched peritoneal exudate cells of mice infected with BCG, as well as from macrophage-like tumor cells (PU5-1.8) and peritoneal macrophages of pretreated mice, which have been propagated in vitro with macrophage growth factor and stimulated with LPS [B. B. Aggarwal et al., J. Biol. Chem. , 260, pp. 2345-54 (1985); D. Mannel et al., "Macrophages as a Source of Tumoricidal Activity (Tumor Necrotizing Factor)," Infect. Immun. , 30, pp. 523-30 (1980)].

Furthermore, human monocytes isolated from the blood of healthy human donors, and stimulated with lymphokines or LPS, produce chemical agents having cytotoxic or cytostatic effects on mouse target cells and human transformed cells which are useful in the compositions of this invention [N. Matthews, "Production of an Anti-tumor Cytotoxin by Human Mono¬ cytes: Comparison of Endotoxin, Interferons and Other Agents as Inducers," Br. J. Cancer, 45, pp. 615-17 (1982); J. Hammerstrφm, "Soluble Cytostatic Factor(s) Released from Human Monocytes: I. Pro¬ duction and Effect on Normal and Transformed Human Target Cells," Scand. J. Immunol., 15, pp. 311-18 (1982)]. Also useful is a fraction of the c.,-α₂ globulins from the serum of normal humans which frac¬ tion has been shown to be toxic to tumors in mice and to inhibit the growth in vitro of human colon cancer, melanoma and neuroblastoma cell lines [United States patent 4,309,418; S. Green et al., Cancer

Letters, 6_, pp. 235-40 (1979); J. Cell. Biol., l , p. 67 (1978)]. 1

-8-

These natural animal and human TNFs have been subsequently purified to some extent and par¬ tially characterized. [See, for example, United States patent 4,309,418; S. Green^" et al., "Partial Purification Of A Serum Factor That Causes Necrosis Of Tumors," Proc. Nat. Acad. Sci. USA, 73, p. 381 (1976).]

TNFs useful in the combinations and treat¬ ments of this invention may also be produced and purified in large amounts using recombinant DNA tech¬ nology [L. Fransen et al., "Molecular Cloning Of Mouse Tumour Necrosis Factor cDNA And Its Eukaryotic Expres¬ sion," Nucl. Acid Res. , 13, pp. 4417 et seq. (1985); A. L. Marmenout et al., "Molecular Cloning And Expres- sion Of Human Tumor Necrosis Factor And Comparison With Mouse Tumor Necrosis Factor," Eur. J. Biochem, 152, pp. 515-22 (1985); see also D. Pennica et al., Nature, 312, pp. 724-28 (1984); T. Shirai, Nature, 313, pp. 803-06 (1985); A. M. Wang et al., Science, 228, pp. 149-54 (1985)].

Without being bound by theory, we believe that the enhanced growth inhibition or killing effect of our combinations and treatments, over those charac¬ terized by TNF alone, is due to inhibition of the mitochondral protein synthesis by the broad spectrum antibiotics, preferably chloramphenicol and tetra¬ cycline, used in accordance with this invention in combination with those TNFs. The mitochondrion has been suggested to be an intracellular target of TNF- mediated effects [N. Matthews, "Anti-tumour cyto- toxin produced by human monocytes: Studies on its mode of action," Br. J. Cancer, 48, pp. 405-10 (1983). Kilbourn et al. (preprint)]. Accordingly, TNF's attack on this intracellular organelle of tumor cells may be enhanced by the antibiotic inhibition of the mitochondria's protein synthesis. -9-

The combinations and treatments of this invention are useful in treating any mammal, including humans. TNFs derived from the target species are preferably used. However, TNFs derived from other species may be used in the combinations and treatments of this invention if they are active in the target cells. For example, mouse TNF has been shown to be active in human cell lines in vitro.

According to this invention, mammals are treated with pharmaceutically effective amounts of the two active components — TNF and a broad spectrum antibiotic — of the combinations of this invention for a period of time sufficient to suppress tumor growth, and preferably to kill tumor cells. The mammals are treated with a composition comprising a combination of TNF and a broad spectrum antibiotic which impedes or inhibits ribosomal protein synthesis. Preferably they are treated sequentially with the two components. However, the particular sequence of treatment chosen does not appear to be important.

More specifically, mammals may be treated with sub¬ cutaneous, intravenous or intramuscular injections of TNF of between about 10 μg to 100 mg per patient per day. However, this dosage should be adjusted by the treating physician according to recognized medical standards, to accommodate the physical condition and acceptance level of the patient. In accordance with this invention, they are also treated with a pharmaceutically effective amount of a broad spectrum antibiotic before, concurrently, of after treatment with TNF. Most commonly, the antibiotic is administered orally, its dosage being the standard one used against infection. [See Goodman and Gilman's, supra, pp. 1185, 1193 and 1222-27.] -10-

In order that the invention described herein may be more fully understood, the following example is set forth. It should be understood that this example is for illustrative purposes only, and is not to be construed as limiting this invention in any manner.

Example

Table I depicts the use of the combinations of this invention. More particularly, it shows the effect of recombinant TNF in combination with chlor¬ amphenicol, thioamphenicol (a more water-soluble and less toxic derivative of chloramphenicol) and tetra¬ cycline, on the B16B16, PG19 and Friend leukemia 745 cells.

-11-

TABLE I cell line μg/ml 50% growth 25% growth inhibition at: inhibition

U/ml r-hTNF U/ml r-hTNF

CHLORAMPHENICOL

B16B16 0 >1.5xl0⁴ >1.5xl0⁴

2 >1.5xl0⁴ >1.5xl0⁴

6 960 320

19 320 180

56 320 100

PG19 0 >1.5xl0⁴ l.lxlO³

12 640 240

37 370 120

110 370 120

Friend leukemia 0 >1.5xl0⁴ 1.5xl0⁴

745 19 >1.5xl0⁴ 600

56 >1.5xl0⁴ 600

160 >1.5xl0⁴ 960

THIOAMPHENICOL

PG19 0 >1.5xl0⁴ 3.3 10³

40 l.lxlO³ 210

120 640 120

370 640 120

TETRACYCLINE

PG19 0 >1.5 10⁴ 640

12 1.1 10³ 370

37 210 120

110 120 70 Our growth inhibition assay quantified the amount of TNF needed to reduce the growth rate of the target cell by 50% or 25% in the presence or absence of antibiotic. More than 1.5 x 10 4 units/ml of recombinant h-TNF alone are needed to reduce cell growth 50% or 25% in a 3-day growth assay, as depicted -12- in Table I. However, we observed a drastic decrease in the amount of TNF needed to reduce cell growth A-l. For example, in our assay with B16B16 cells, the amount σ_rr-TNF needed to inhibit growth 50%

4 dropped from more than 1.5 x 10 U/ml to 320 U/ml when we added 19 micrograms/ml chloramphenical.

Especially dramatic, in our assay with PG19 cells, the amount of r-TNF needed to inhibit growth 50% dropped from more than 1.5 x 10 4 U/ml to 640 U/ml when we added tetracycline in concentration of 37 micrograms/ml. We also observed positive results in our assay using Friend leukemia 745 cells. Our results with Friend leukemia 3C18 cells (an IFN α/β resistant subline of 745) were similar to the results we obtained with 745 cell line.

While we have hereinbefore presented a number of embodiments of this invention, it is apparent that our basic construction can be altered to provide other embodiments which utilize the processes and compositions of this invention. There¬ fore, it will be appreciated that the scope of this invention is to be defined by the claims appended hereto rather than by the specific embodiments which have been presented hereinbefore by way of example.

Claims

-13-CLAIMS

1. A combination of TNF and an antibiotic which inhibits or impedes protein synthesis in pharma¬ ceutically effective amounts.

2. The combination according to claim 1, wherein the antibiotic is selected from the group consisting of chloramphenicol and tetracycline.

3. The combination according to claim 1, wherein the amount TNF is between about 10 μg and 100 mg.

4. The combination according to claim 1, wherein the TNF is recombinant TNF.

5. A method of treating a tumor-bearing mammal comprising the steps of administering to the mammal, pharmaceutically effective amounts of TNF and pharmaceutically effective amounts of an anti¬ biotic which inhibits or impedes protein synthesis.

6. The method according to claim 5, characterized in that the antibiotic is selected from the group consisting of chloramphenicol and tetracyline.

7. The method according to claim 5, wherein the amount of TNF is between about lOμg and 100 mg.

8. The method according to claim 5, wherein the TNF is recombinant TNF.

9. The use of pharmaceutically effective amounts of TNF and pharmaceutically effective amounts of an antibiotic which inhibits or impedes protein

-14- synthesis, for the production of a pharmaceutically acceptable combination for the treatment of tumors.

10. The use according to claim 9, charac¬ terized in that the antibiotic is selected from the group consisting of chloramphenicol and tetracycline.

11. The use according to claim 9, wherein the amount of TNF is between about lOμg and 100 mg.

12. The use according to claim 9, wherein the TNF is recombinant TNF.