Combinations of tumor necrosis factors and anti-inflammatory agents and methods for treating malignant and non-malignant diseases

This invention relates to combinations and methods for the treatment of malignant and non-malignant diseases. More particularly, this invention relates to combinations of natural or recombinant tumor necrosis factors ("TNF") and non-steroidal anti-inflammatory agents, such as indomethacin and ibuprofen, useful for the growth inhibition or killing of transformed cells. According to this invention, the non-steroidal anti-inflammatory agents are used to reduce or eliminate the toxic side effects of high doses of TNFs employed in the treatment of malignant and non-malignant neoplastic diseases. Advantageously, the combinations and methods of this invention allow the administration of higher doses of TNF than those tolerated in conventional treatment regimens based upon TNF alone.

TECHNICAL FIELD OF INVENTION 
This invention relates to combinations and methods for the treatment of 
malignant and non-malignant diseases. More particularly, this invention 
relates to combinations of natural or recombinant tumor necrosis factors 
("TNF") and non-steroidal anti-inflammatory agents, such as indomethacin 
and ibuprofen, useful for the growth inhibition or killing of transformed 
cells. According to this invention, the non-steroidal anti-inflammatory 
agents are used to reduce or eliminate the toxic side effects of high 
doses of TNFs employed in the treatment of malignant and non-malignant 
neoplastic diseases. Advantageously, the combinations and methods of this 
invention allow the administration of higher doses of TNF than those 
tolerated in conventional treatment regimens based upon TNF alone. 
BACKGROUND ART 
TNF is a protein produced by macrophages and mononuclear phagocytes upon 
activation by endotoxin or other microbial products or stimuli [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); P. J. 
Hotez et al., "Lipoprotein Lipase Suppression In 3T3-L1 Cells By A 
Haematoprotozoan-Induced Mediator From Peritoneal Exudate Cells", Parasite 
Immunol., 6, pp. 203-09 (1984)]. Although TNF 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, it exerts little or no cytotoxicity on normal cells [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 Search For Specificity - G.H.A. 
Clowes Memorial Lecture", Cancer Research, 41, pp. 361-75 (1981); B. D. 
Williamson et al., Proc. Natl. Acad. Sci. USA, 80, pp. 5397-401 (1983)]. 
Malignant diseases are a group of diseases characterized by tumorigenic or 
neoplastic cell growth. Such diseases include malignant hematological 
systemic diseases, carcinomas, sarcomas, myelomas, melanomas, lymphomas 
and papillomas. Non-malignant neoplastic diseases, including non-malignant 
tumors, are also characterized by neoplastic cell growth which is 
localized to a specific area. The transformation of normal cells within 
the body into either malignant or non-malignant neoplasms may be induced 
by chemical carcinogens, radiation, physical agents or spontaneous 
tumorigenic growth. 
The precise etiology of many malignant and non-malignant diseases remains 
unknown. Accordingly, treatments for these diseases are limited, and 
effective agents are not always conventionally available for a specific 
disease. Such diseases have been treated, for example, by surgical 
techniques or by non-surgical methods including chemotherapy, radiation 
and immunotherapy. Any value of such treatment techniques, however, is 
often diminished by adverse side effects or risks attendant with their 
use. For example, non-surgical techniques such as chemotherapy generally 
have immunosuppressant effects and may increase the patient's 
susceptibility to secondary infections. Surgical treatments to excise 
malignant or non-malignant tumors involve risks which accompany any 
invasive procedure and may not effectively remove or eliminate the entire 
transformed cell population. Moreover, certain malignant diseases are 
resistant to conventional treatment techniques. For example, most skin 
melanomas are considered to be radio-resistant. No single agent or 
combination chemotherapy has been successful in effecting consistent 
regressions of malignant melanomas. Malignant renal cell carcinoma is also 
resistant to available single agent and combination chemotherapies. 
Alternative methods of treatment for malignant and non-malignant diseases 
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 administrations of the murine antibody [G. E. 
Goodman et al., "Pilot Trial Of Murine Monoclonal Antibodies In Patients 
With Advanced Melanoma", J. Clin. Oncol., 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 therapies, various treatments have 
been directed to augmenting the body's immune response to tumorigenic 
cells by increasing the body's level of certain 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]. Combinations 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., 
"Recombinant Tumor Necrosis Factor: Its Effect And Its Synergism With 
Interferon-.gamma. On A Variety Of Normal And Transformed Human And Mouse 
Cell Lines", Eur. J. Cancer Clin. Oncol., 22, pp. 419-26 (1986); B. D. 
Williamson et al., "Human Tumor Necrosis Factor Produced By Human B-Cell 
Lines: Synergistic Cytotoxic Interaction With Human Interferon", Proc. 
Natl. Acad. Sci. USA, 80, pp. 5397-401 (1983); see also European patent 
application 131,789]. Although TNF has shown promise as a potent cytotoxic 
agent, its usefulness as a therapeutic for treating malignant and 
non-malignant diseases has been restricted by dose-limiting toxic side 
effects. 
TNF has been suggested as one of the mediators in the pathogenesis of 
endotoxic shock [B. Beutler et al., "Passive Immunization Against 
Cachectin/Tumor Necrosis Factor Protects Mice From Lethal Effect Of 
Endotoxin", Science, 229, pp. 869-71 (1985); B. Beutler and A. C. Cerami, 
"Cachectin And Tumor Necrosis Factor As Two Sides Of The Same Biological 
Coin", Nature, 320, pp. 584-88 (1986)]. In addition to its contribution to 
such systemic effects, TNF can play a role in local inflammation as in 
osteoarthritis [J. M. Dayer et al., "Cachectin/ Tumor Necrosis Factor 
Stimulates Collagenase And Prostaglandin E.sub.2 Production By Human 
Synovial Cells And Dermal Fibroblasts", J. Exp. Med., 162, pp. 2163-68 
(1985)]. 
The role of TNF in such pathogeneses may be attributable to its stimulation 
of prostaglandin or thromboxane production. Although there is no clear 
explanation of the pathogenic mechanisms in toxic shock, substantial 
increases in circulating prostaglandins have also been reported in a 
variety of experimental models for hemorrhagic and endotoxic shock and the 
thromboxane PGI.sub.2, as well as the prostaglandin PGE.sub.2, have been 
proposed as important mediators in the development of irreversible shock 
[J. R. Fletcher, in Biological Protection With Prostaglandins, I, pp. 
65-72 (1985); R. R. Butler et al., "Elevated Plasma Levels Of Thromboxane 
(Tx) and Prostacyclin (PGI.sub.2) In Septic Shock", Circ. Shock, 8, pp. 
213-14 (1981); R. H. Demling et al., Am. J. Physiol., 240, pp. H348-53 
(1981); W. C. Wise et al., "Implications For Thromboxane A.sub.2 In The 
Pathogenesis Of Endotoxic Shock", Adv. Shock Res., 6, p. 83 (1981); H. 
Bult et al., "Blood Levels Of 6-Keto-PGF.sub.1.alpha., The Stable 
Metabolite Of Prostacyclin During Endotoxin-Induced Hypotension", Arch. 
Int. Pharmacodyn, 236, pp. 285-86 (1978); J. A. Cook et al., "Elevated 
Thromboxane Levels In The Rat During Endotoxic Shock", J. Clin. Invest., 
65, pp. 227-30 (1980)]. Although it has been reported that non-steroidal 
anti-inflammatory drugs appear to protect against certain lethal effects 
of endotoxin in experimental animals, such agents have not been used 
clinically to treat human patients in endotoxic and hemorrhagic shock [B. 
L. Short et al., "Indomethacin Improves Survival In Gram-Negative Sepsis", 
Adv. Shock Res., 6, pp. 27-36 (1981); P. M. Almqvist et al., "Treatment Of 
Experimental Canine Endotoxin Shock With Ibuprofen, A Cyclooxygenase 
Inhibitor", Circ. Shock, 131, pp. 227-32 (1984); E. R. Jacobs, J. Clin. 
Invest., 70, pp. 536-41 (1982) P. V. Halushka et al., "Protective Effects 
Of Aspirin In Endotoxic Shock," J. Pharmacol. Exp. Therm., 218, pp. 464-69 
(1981)]. 
To date, therefore, conventional methods and therapeutic agents have not 
proved to be effective in the treatment of many malignant and 
non-malignant diseases. Accordingly, the need exists for therapeutic 
agents and methods which avoid the disadvantages of these conventional 
agents and methods while providing effective treatment for these diseases. 
DISCLOSURE OF THE INVENTION 
The present invention solves the problems referred to above by providing 
effective combinations and methods for the treatment of malignant and 
non-malignant diseases. According to this invention, natural or 
recombinant tumor necrosis factors ("TNFs") are used in combination with 
non-steroidal anti-inflammatory agents for treating malignant and 
non-malignant neoplastic diseases. Advantageously, the combinations and 
methods of this invention prevent or reduce the potential side effects of 
treatments with high dosages of TNF alone, while not interfering with the 
cytotoxic activity of TNF against undesirable malignant or non-malignant 
cell proliferations.

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. 
In the description, the following terms are employed: 
TNF (or tumor necrosis factor)--TNF is a growth inhibitory or cytotoxic 
monokine. Natural TNF is a protein with a subunit 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. Marmenout et 
al., "Molecular Cloning And Expression Of Human Tumor Necrosis Factor And 
Comparison With Mouse Tumor Necrosis Factor", Eur. J. Biochem., 152, pp. 
515-22 (1985); L. Fransen et al., "Molecular Cloning Of Mouse Tumor 
Necrosis Factor cDNA And Its Eukaryotic Expression", Nucl. Acids Res., 13, 
pp. 4417 et seq. (1985); see also D. Pennica et al., "Human Tumour 
Necrosis Factor: Precursor Structure, Expression, And Homology To 
Lymphotoxin", Nature, 312, pp. 724-29 (1984); T. Shirai, "Cloning And 
Expression In Escherichia Coli Of The Gene For Human Tumour Necrosis 
Factor", Nature, 313, pp. 803-06 (1985); A. M. Wang et al., "Molecular 
Cloning Of The Complementary DNA For Human Tumor Necrosis Factor", 
Science, 228, pp. 149-54 (1985)]. 
The nucleotide sequence of cloned TNF indicates 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 or cytotoxic activity of these TNFs. They include TNF-like 
compounds from a variety of sources, such as natural TNFs, recombinant 
TNFs, and synthetic or semi-synthetic TNFs. 
As used in this application, "TNF" also includes the closely-related 
polypeptide lymphotoxin, also known as TNF-.beta.. [D. Pennica et al., 
supra, Nature, 312, pp. 724-29; P. W. Gray et al., "Cloning And Expression 
Of cDNA For Human Lymphotoxin, A Lymphokine With Tumour Necrosis 
Activity", Nature, 312, pp. 721-24 (1984); B. Y. Rubin et al., 
"Purification And Characterization Of A Human Tumor Necrosis Factor From 
The LukII Cell Line," Proc. Natl. Acad. Sci. USA, 82, pp. 6637-41 (1985).] 
Malignant Disease--Any disease characterized by tumorigenic or neoplastic 
cell growth, including malignant hematological systemic diseases, 
carcinomas, sarcomas, myelomas, melanomas, leukemias, lymphomas and 
papillomas. 
Non-Malignant Neoplastic Disease--Any disease characterized by an 
undesirable proliferation of cells which is localized to the site of 
origin, such as benign growths. 
This invention relates to combinations and methods for treating malignant 
and non-malignant neoplastic diseases. More particularly, this invention 
relates to combinations of pharmaceutically effective amounts of TNF and 
pharmaceutically effective amounts of non-steroidal anti-inflammatory 
agents that block the side effects of high dosages of TNF. Such side 
effects include hypothermia, metabolic acidosis, hypoglycemia, peripheral 
cyanosis, diarrhea and other effects similar to those seen in endotoxic 
shock. According to one embodiment, the method of this invention comprises 
the step of treating a mammal in a pharmaceutically acceptable manner with 
a pharmaceutically effective amount of TNF and a pharmaceutically 
effective amount of a compound selected from the group consisting of 
non-steroidal anti-inflammatory agents for a period of time sufficient to 
exert cytotoxic or cytostatic effects against the tumor or other 
neoplastic cell population. 
Among the TNFs useful in the combinations and methods 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 and rabbits which have been infected 
with Bacillus-Calmette-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. Immunol., 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 Monocytes: 
Comparison Of Endotoxin, Interferons And Other Agents As Inducers", Br. J. 
Cancer, 45, pp. 615-17 (1982); J. Hammerstr.phi.m, "Soluble Cytostatic 
Factor(s) Released From Human Monocytes: I. Production And Effect On 
Normal And Transformed Human Target Cells", Scand. J. Immunol., 15, pp. 
311-18 (1982)]. Also useful is a fraction of the .alpha..sub.1 
-.alpha..sub.2 globulins from the serum of normal humans 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., 79, p. 67 (1978)]. 
These natural animal and human TNFs have been subsequently purified to some 
extent and partially characterized. [See, for example, U.S. Pat. No. 
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 methods of this invention may also be 
produced and purified in large amounts using recombinant DNA technology 
[L. Fransen et al., "Molecular Cloning Of Mouse Tumour Necrosis Factor 
cDNA And Its Eukaryotic Expression", Nucl. Acids Res., 13, pp. 4417 et 
seq. (1985); A. L. Marmenout et al., "Molecular Cloning And Expression 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)]. 
The anti-inflammatory agents useful in the combinations and methods of this 
invention include non-steroidal anti-inflammatory agents that are also 
cyclooxygenase inhibitors, which inhibit the biosynthesis of 
prostaglandins, prostacyclins or thromboxanes. Such agents inhibit the 
arachidonic acid cyclooxygenase, which is also known as prostaglandin 
synthetase. These non-steroidal anti-inflammatory agents include, but are 
not limited to, acetyl salicylic acid (aspirin), methyl salicylate, sodium 
salicylate, phenylbutazone, oxyphenbutazone, apazone, indomethacin, 
sulindac, tolmetin, mefenamic acid, ibuprofen, naproxen, fenoprofen, 
flurbiprofen, ketoprofen and other compounds having a similar ability to 
block prostaglandin, prostacyclin or thromboxane synthesis. Other 
anti-inflammatory agents useful in the combinations and methods of this 
invention are lipocortins derived from natural sources or lipocortins and 
lipocortin-like polypeptides produced by recombinant techniques [see U.S. 
Pat. application Ser. Nos. 690,146; 712,376; 765,877 and 772,892; B. 
Wallner et al., "Cloning And Expression Of Human Lipocortin A 
Phospholipase A-2 Inhibitor With Potential Anti-Inflammatory Activity", 
Nature, 320, pp. 77-81 (1986)]and uromodulin [A. V. Muchmore and Jean M. 
Decker, "Uromodulin: A Unique 85-Kilodalton Immunosuppressive Glycoprotein 
Isolated From Urine Of Pregnant Women", Science, 229, pp. 479-81 (1985)]. 
The combinations and methods of the present invention allow the 
administration of TNF in higher doses than those tolerated in conventional 
treatment regimens based upon TNF alone. Accordingly, the combinations and 
methods of this invention advantageously reduce or eliminate the toxic 
effects of high dose treatments with TNF alone. Without being bound by 
theory, we believe that the effectiveness of our combinations and methods 
over those using TNF alone is due to the action of the anti-inflammatory 
agents in blocking the production of one or more of the prostaglandins, 
prostacyclins or thromboxanes in the body resulting from high dosages of 
TNF, thereby reducing the toxicity of TNF. This allows the administration 
of TNF in high doses formerly typically accompanied by toxic effects. 
Thus, the use of TNF in combination with a non-steroidal anti-inflammatory 
agent may reduce the duration and level of treatment which would be 
required by therapies based upon conventionally tolerated lower dosages of 
TNF alone. 
The combinations and methods of this invention are useful in treating any 
mammal, including humans. TNFs derived from the target patient species are 
preferably used. However, TNFs derived from other species may be used in 
the combinations and methods 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 [L. Fransen et al., "Recombinant Tumor Necrosis 
Factor: Species Specificity For A Variety Of Human And Murine Transformed 
Cell Lines", Cell. Immunol., 100, pp. 260-67 (1986)]. 
According to this invention, mammals are treated with pharmaceutically 
effective amounts of the two active components--TNF and a non-steroidal 
anti-inflammatory agent--of the combinations of this invention for a 
period of time sufficient to inhibit malignant or undesirable 
non-malignant cell proliferation, e.g., suppress tumor or neoplastic cell 
growth, and preferably to kill tumor or neoplastic cells. 
In accordance with this invention, pharmaceutically effective amounts of a 
non-steroidal anti-inflammatory agent and the TNF are administered 
sequentially or concurrently to the patient. However, the particular 
sequence of treatment does not appear to be important. The most effective 
mode of administration and dosage regimen of TNF and anti-inflammatory 
agent will depend upon the type of disease to be treated, the severity and 
course of that disease, previous therapy, the patient's health status and 
response to TNF and the judgment of the treating physician. TNF may be 
administered to the patient at one time or over a series of treatments. 
Preferably, the non-steroidal anti-inflammatory agent and the TNF are 
administered sequentially to the patient, with the non-steroidal 
anti-inflammatory agent being administered before, after, or both before 
and after treatment with TNF. Sequential administration involves treatment 
with the non-steroidal anti-inflammatory agent at least on the same day 
(within 24 hours) of treatment with TNF and may involve continued 
treatment with the anti-inflammatory agent on days that the TNF is not 
administered. Conventional modes of administration and standard dosage 
regimens of anti-inflammatory agents may be used. [See A. G. Gilman et al. 
(Eds.), The Pharmacological Basis Of Therapeutics, pp. 697-713, 1482, 
1489-91 (1980); Physicians Desk Reference, (1986 Edition).] For example, 
indomethacin may be administered orally at a dosage of about 25-50 mg, 
three times a day. Higher doses may also be used. Alternatively, aspirin 
(about 1500-2000 mg/day), ibuprofen (about 1200-3200 mg/day), or 
conventional therapeutic doses of other non-steroidal anti-inflammatory 
agents may be used. Dosages of non-steroidal anti-inflammatory agents may 
be titrated to the individual patient. 
According to one embodiment of this invention, the patient may receive 
concurrent treatments with the non-steroidal anti-inflammatory agent and 
TNF. Local, intralesional or intravenous injection of TNF is preferred. 
[See A. G. Gilman et al., supra at pp. 1290-91.] The non-steroidal 
anti-inflammatory agent should preferably be administered by subcutaneous 
injection or orally. 
Alternatively, the patient may receive a composition comprising a 
combination of TNF and a non-steroidal anti-inflammatory agent according 
to conventional modes of administration of agents which exhibit 
anticancer, antitumor or anti-inflammatory activity. These include, for 
example, parenteral, subcutaneous, intravenous or intralesional routes of 
administration. 
The compositions used in these therapies may also be in a variety of forms. 
These include, for example, solid, semi-solid and liquid dosage forms, 
such as tablets, pills, powders, liquid solutions or suspensions, 
liposomes, suppositories, injectable and infusable solutions. The 
preferred form depends on the intended mode of administration and 
therapeutic application. The compositions also preferably include 
conventional pharmaceutically acceptable carriers and adjuvants which are 
known to those of skill in the art. Preferably, the compositions of the 
invention are in the form of a unit dose and will usually be administered 
to the patient one or more times a day. 
TNF may be administered to the patient in any pharmaceutically acceptable 
dosage form including intravenous, intramuscular, intralesional or 
subcutaneous injection. An effective dose may be in the range of from 
about 0.01 to about 1.0 mg/kg body weight, it being recognized that lower 
and higher doses may also be useful. More particularly, doses of TNF 
higher than those typically tolerated in patients treated with TNF alone 
may advantageously be used in the methods and compositions of this 
invention. 
It should, of course, be understood that the compositions and methods of 
this invention may be used in combination with other cancer or tumor 
therapies, e.g., interferons (e.g., IFN-.alpha., IFN-.beta. and 
IFN-.gamma.) or chemotherapy, for the treatment of malignant and 
non-malignant diseases in mammals. 
Once improvement of the patient's condition has occurred, a maintenance 
dose is administered if necessary. Subsequently, the dosage or the 
frequency of administration, or both, may be reduced, as a function of the 
symptoms, to a level at which the improved condition is retained. When the 
symptoms have been alleviated to the desired level, treatment should 
cease. Patients may, however, require intermittent treatment on a 
long-term basis upon any recurrence of disease symptoms. 
In order that the invention described herein may be more fully understood, 
the following examples are set forth. It should be understood that these 
examples are for illustrative purposes only, and are not to be construed 
as limiting this invention in any manner. 
EXAMPLE 1 
This example represents the in vivo action of non-steroidal 
anti-inflammatory agents in blocking the toxic side effects of treatment 
with high doses of TNF. In this example, we administered TNF at a dosage 
which was lethal when given by an intravenous route, due to 
life-threatening side effects such as hypothermia, metabolic acidosis, 
hypoglycemia and peripheral cyanosis. This dose would have been well 
tolerated if given subcutaneously, because blood levels of TNF attained 
via that route are never as high as those resulting from intravenous 
administration. 
The data set forth below show that much higher blood levels of TNF are 
tolerated, without side effects, when TNF is administered in combination 
with a non-steroidal anti-inflammatory agent rather than alone. 
Accordingly, this example demonstrates that treatment with a non-steroidal 
anti-inflammatory agent concurrently with the administration of TNF blocks 
any adverse physiological responses induced by high blood levels of TNF. 
The methods and compositions of this invention, therefore, advantageously 
enhance the usefulness of TNF, particularly at high doses formerly 
typically associated with undesirable side effects, as a therapeutic agent 
for treating malignant and non-malignant neoplastic diseases. 
In this example, male rats (CD strain, Charles River Breeding Laboratories, 
Wilmington, Mass.) weighing 50-60 grams each, were divided into six 
treatment groups. All the rats were maintained on a standard diet of 
Purina Rat Chow and water for 3 days prior to treatment. We treated each 
of Groups 1-5 with either TNF alone, an anti-inflammatory agent alone, or 
TNF in combination with an anti-inflammatory agent, as indicated below. 
Group 6 served as a vehicle control group. 
Group 1: 4 .mu.g/g body weight human recombinant TNF intravenously. 
Group 2: 3 mg/kg body weight indomethacin intraperitoneally; followed by 4 
.mu.g/g body weight human recombinant TNF intravenously 2 hours later. 
Group 3: 3 mg/kg body weight indomethacin intraperitoneally; phosphate 
buffered saline intravenously 2 hours later. 
Group 4: 20 mg/kg body weight ibuprofen intraperitoneally; followed by 4 
.mu.g/g body weight human recombinant TNF intravenously 2 hours later. 
Group 5: 20 mg/kg body weight ibuprofen intraperitoneally; phosphate 
buffered saline intravenously 2 hours later. 
Group 6: vehicle control (-) phosphate buffered saline intraperitoneally 
and intravenously. 
The TNF used in this example was recombinant human TNF, supplied by Biogent 
(Ghent, Belgium) and Biogen Inc. (Cambridge, Mass.). The preparation was 
more than 99% pure, contained less than 20 ng/mg endotoxin and had a 
specific activity in the range of about 9.6.times.10.sup.6 units/mg to 
2.5.times.10.sup.7 units/mg. The indomethacin and ibuprofen were supplied 
by Sigma Co. and Upjohn Co., respectively. 
TNF was administered by intravenous injection into the jugular vein 
performed under ether anesthesia. We collected blood samples by puncture 
of the jugular vein before and at hourly intervals after TNF or phosphate 
buffered saline injection. We then centrifuged the collected blood in 
heparinized tubes and stored the plasma at -20.degree. C. We measured 
plasma glucose levels using a Beckman Glucose Analyzer (Beckman 
Instruments Co.). Prostaglandin metabolite levels were measured by 
radioimmunoassay as described in L. Levine, Biochem. of Arach. Acid 
Metab., pp. 405-16 (1985). Rectal temperatures were measured using an 
electronic thermometer (Model 49TA, Yellow Springs Instrument Co., Inc.). 
All results are expressed as Means .+-.SEM and the statistical 
significance of changes seen were evaluated with the Unpaired Student's t 
test. 
Effect Of TNF Treatment Alone 
As demonstrated in FIG. 1A, among the Group 1 rats, no deaths occurred 
during the first hour post-TNF injection. After that time, however, a 
progressive loss in viability was observed, with death of all the animals 
typically resulting within a period of 2 to 4 hours. For example, by 2 
hours post-injection, about 25% of the animals had died and by 4 hours 
post-injection, all of the Group 1 rats were dead. In total, 35 animals 
were treated in this fashion, and 30 died this rapidly. 
The rapid decrease in viability of the Group 1 rats was accompanied by a 
spectrum of physiological changes resembling those observed in studies of 
experimentally-induced endotoxic shock [J. P. Filkins, Am. J. Emerg. Med., 
2, pp. 70-73 (1984) J. P. Filkins, Fed. Proc., pp. 300-04 (1985)]. These 
physiological changes included hypothermia, peripheral cyanosis, metabolic 
acidosis, diarrhea, initial hyperglycemia followed by severe hypoglycemia, 
and increased prostaglandin synthesis. 
As demonstrated in FIG. 1B, TNF induced a sharp decrease in body 
temperature within 1 hour post-injection, with the mean rectal temperature 
of the treated animals falling from 36.8.degree. C. to 34.3.degree. C. 
This decrease in temperature clearly preceded other symptoms and, 
therefore, appeared to be a specific physiological effect of TNF, rather 
than a consequence of the loss of viability. Between 3 and 4 hours 
post-injection, body temperatures continued to decrease to between 
29.5.degree. C. and 32.degree. C. This hypothermic effect is surprising, 
since lower intravenous doses of TNF, or the equivalent amount of TNF 
injected subcutaneously, induced a fever which was blocked with 
cyclooxygenase inhibitors. 
Other physiological changes accompanied the hypothermia. For example, in 
the final hour before death, the animals appeared very lethargic and 
showed cyanosis in their extremities. In addition, at about 1 hour 
post-injection, the animals had diarrhea. Upon postmortem examination, the 
intestines of the TNF-treated rats appeared empty, even though the animals 
had free access to food prior to treatment. In contrast, the intestines of 
the control rats or of control rats deprived of food for 4 hours were full 
of solid material. 
Another result of the TNF treatment was initial hyperglycemia, followed by 
severe hypoglycemia. As demonstrated in FIG. 2, large biphasic changes in 
blood glucose resulted after the TNF injections. Initially, the rats 
developed hyperglycemia which, within one hour, was followed by a sharp 
decrease in plasma glucose levels to about 30 mg/ 100 ml (1.6mM). Four 
hours after the TNF injection, when most of the animals were near death, 
their blood glucose levels had fallen further to about 20 mg/ 100 ml. Such 
levels, if maintained, are not generally sufficient for survival. 
The TNF-treated rats also exhibited increased prostaglandin levels in their 
blood serum. By determining the blood content of the stable metabolite of 
PGE.sub.2, 13,14-dihydro-l15-keto-PGE2 ("DHK-PG"), we measured body 
prostaglandin production after TNF treatment. Within 1 hour 
post-injection, TNF induced a substantial increase in PGE.sub.2 
production. As shown in Table 1, plasma levels of DHK-PG increased 
ten-fold, from 0.40.+-.0.05 ng/ml to 4.26.+-.0.48 ng/ml. The high levels 
of this metabolite of PGE.sub.2 were maintained for several hours, and at 
3 hours post-injection, reached 5.77.+-.0.51 ng/ml. These increases were 
unexpected since prostaglandins and, in particular, PGE.sub.2, have been 
associated with the induction of fever rather than hypothermia [H. A. 
Bernheim et al., J. Physiol., 301, pp. 69-78 (1980)]. 
In treatments similar to those described above for Groups 1-3 and 6, we 
observed that TNF treatment also resulted in severe metabolic acidosis. 
As shown in Table 2, we measured blood pH, pCO.sub.2 (mm Hg) and HCO.sub.3 
(.mu.mol/1) in four separate groups of rats treated as follows: 
Group A (4 rats): vehicle control-phosphate buffered saline intravenously. 
Group B (8 rats): 4 .mu.g/g body weight human recombinant TNF 
intravenously. 
Group C (7 rats): 3 mg/kg body weight indomethacin intraperitoneally; 
followed by 4 .mu.g/g body weight human recombinant TNF intravenously 2 
hours later. 
Group D (5 rats): 3 mg/kg body weight indomethacin intraperitoneally; 
followed by phosphate buffered saline intravenously 2 hours later. 
We collected arterial blood from the abdominal aorta under ether anesthesia 
in heparinized syringes 3 hours after TNF or saline injection. We measured 
blood pH and pCO.sub.2 (mm Hg) in a Blood Gas Analyzer. HCO.sub.3 
(.mu.mol/1) was measured using the Henderson-Hasselbach Equation. As shown 
in Table 2, the arterial pH of rats treated with TNF alone fell 
significantly, their pCO.sub.2 decreased by about 30%, and their arterial 
bicarbonate (HCO.sub.3) concentration was about half that in the controls. 
The physiological effects resulting from the TNF treatment are typical of 
those observed in animals with endotoxic shock. Accordingly, although 
activated monocytes produce many potent polypeptides, overproduction of 
TNF may by itself account for most of the life-threatening symptoms of 
irreversible shock. 
In order to verify that the various physiological effects observed were not 
due to endotoxin contamination of the TNF solutions themselves, we heated 
the TNF at 70.degree. C. for 15 minutes to destroy TNF activity but not to 
affect any endotoxins [E. A. Carswell et al., supra, p. 1]. Administration 
of the thus-treated TNF to rats did not cause the death of any animal. In 
addition, none of the treated rats exhibited changes in body temperature 
or developed diarrhea. 
Effect Of Combination Non-Steroidal Anti-Inflammatory/TNF Treatments 
The Group 2 and Group 4 rats treated with a single intraperitoneal 
injection of a non-steroidal anti-inflammatory/prostaglandin synthetase 
inhibitor before receiving TNF treatment, did not exhibit the symptoms 
seen in the Group 1 rats. Thus, indomethacin (Group 2) and ibuprofen 
(Group 4) were found to prevent the toxic effects of high dosage levels of 
TNF administration. 
As demonstrated in FIG. 1A, a single injection of indomethacin before TNF 
treatment provided a high degree of protection against the lethal effects 
of TNF. All of the Group 2 rats were alive 4 hours after receiving the TNF 
injection. For example, among eight Group 2 rats, one rat died 12 hours 
after that injection and two more rats died 21 hours after that injection. 
The other five rats in Group 2 remained alive and appeared normal 
subsequently. 
In treatments similar to those described above for Group 1 and Group 2 
rats, we again observed that a single injection of indomethacin provided 
protection against TNF-induced mortality. More specifically, while each of 
a group of 16 rats injected with TNF alone died within 4 hours of 
treatment, each of the group of 20 rats treated with indomethacin prior to 
TNF treatment were alive 4 hours after receiving the TNF injection. Four 
of the indomethacin-treated rats died within the period of 4-6 hours after 
the TNF injection and three more rats died within 6-24 hours after that 
injection. No further deaths occurred within the period of 24-56 hours 
post-TNF injection, at which time 65% of the rats were viable and appeared 
healthy. 
Similarly, we observed that rats who received a single injection of 
indomethacin after TNF treatment were protected against the lethal effects 
of TNF. While animals receiving only TNF all died within 4 hours of 
treatment, rats who were treated with indomethacin one hour after TNF 
administration were alive 6 hours after the TNF injection. 
The Group 4 rats, who received a single injection of ibuprofen before TNF 
treatment were also protected against the lethal effects of TNF. As 
demonstrated in FIG. 3A, 75% of the ibuprofen-treated rats were still 
alive 6 hours after the TNF injection. By 24 hours after TNF treatment, 
55% of the rats were viable and appeared healthy. 
We believe that repeated administration of indomethacin or ibuprofen in the 
treatments described above would have further reduced any TNF induced 
mortality. 
Both indomethacin and ibuprofen prevented the rapid decrease in body 
temperature and the subsequent progressive hypothermia seen in animals 
treated with TNF alone. As demonstrated in FIGS. 1B and 3B, several of the 
rats treated with indomethacin or ibuprofen before TNF treatment showed 
only a slight decrease in body temperature--1.degree. or 2.degree. 
C.--which quickly returned to normal levels. Furthermore, when 
indomethacin was administered to five rats 1 hour after TNF treatment, 
when hypothermia was already evident, their temperatures rose and returned 
to normal. 
The Group 2 (indomethacin-treated) and Group 4 (ibuprofen-treated) rats 
exhibited neither peripheral cyanosis nor diarrhea. 
In addition, administration of indomethacin or ibuprofen before TNF 
treatment completely blocked the large rise in prostaglandin production, 
as reflected in the serum levels of the DHK-PG metabolite of PGE.sub.2, 
which was seen in those rats treated with TNF alone. As demonstrated in 
Table 1, levels of this metabolite were extremely low in the indomethacin 
and ibuprofen-treated rats. By 3 hours after injection of the 
cyclooxygenase inhibitor, although DHK-PG levels were again detectable and 
approached normal values, they were still much lower than DHK-PG levels in 
the rats treated only with TNF. 
The biphasic changes in plasma glucose levels seen in the TNF-treated rats 
of Group 1 were not seen in the rats receiving either an indomethacin or 
an ibuprofen injection prior to TNF treatment. As shown in FIG. 2, the 
Group 2 rats who received indomethacin did not show any significant 
changes in plasma glucose levels. Ibuprofen injection before TNF treatment 
also decreased the changes in blood glucose. Four hours after the TNF 
treatment, those rats who had received ibuprofen had glucose levels which 
were about 40% lower than the untreated control rats. This decrease in 
blood glucose was much smaller than that seen in the Group 1 rats who were 
treated with TNF alone. 
The significant change in blood pH, pCO.sub.2 and HCO.sub.3 levels which 
accompanied TNF treatment alone was not seen in rats treated with 
indomethacin prior to injection with TNF. As shown in Table 2, the 
bicarbonate (HCO.sub.3) levels of rats that received indomethacin prior to 
injection with TNF were 50% higher than animals that received only TNF. 
Three hours after TNF injection, the arterial pCO.sub.2 and pH of the 
indomethacin-treated rats were indistinguishable from those of the control 
rats. 
While the reduction in TNF side effects demonstrated above resulted from a 
single administration of a non-steroidal anti-inflammatory agent, we 
believe that remaining side effects would be further reduced or eliminated 
by repeated administration of that agent over the course of TNF therapy. 
EXAMPLE 2 
In this example, we examined the effect of non-steroidal anti-inflammatory 
agents on the cytotoxic and cytostatic actions of TNF on various 
transformed cell lines. As shown in FIG. 4, the cytostatic action of TNF 
on cultured tumor cells (Hela cells) was not affected by the presence of 
indomethacin at concentrations that should prevent all prostaglandin 
synthesis. 
Hela D98/AH2 cells (a cell line originally obtained from Dr. E. Stanbridge, 
University of California at Irvine) were cultured in Dulbecco's modified 
Eagle's medium (DMEM) containing 10% fetal calf serum and 25 .mu.g/ml 
gentamycin at 37.degree. C. under 5% CO.sub.2, as described in L. Fransen 
et al., Europ. J. Cancer Clin. Oncol., 22, 419-26 (1986). We plated 
10.sup.4 cells/0.2ml/well in standard microtiter plates containing various 
concentrations of TNF or TNF and indomethacin. TNF was tested at 
concentrations of 3000 units/ml and 1/3 dilutions down to 1 unit/ml. At 
each TNF concentration, indomethacin was tested at concentrations of 
5.times.10.sup.-5 M (a concentration which completely blocks prostaglandin 
synthesis) and 1/3 dilutions down to 5.times.10.sup.-7 M. After 3 days, we 
removed the cell supernatant and quantitated the remaining cells by 
staining them for 10 minutes with a solution of 0.5% crystal violet, 8% 
(V/V) formaldehyde (40%), 0.17% NaCl and 22.3% (V/V) ethanol. The wells 
were then thoroughly washed with tap water, and the bound dye was 
dissolved in 33% acetic acid (0.1 ml/well). The released dye was measured 
spectrophotometrically at 577 nm (Kontron spectrophotometer SLT 210). The 
results are shown in FIG. 4, in which increasing cytotoxicity corresponds 
to decreasing OD-577 values. 
Similar results were observed on the mouse fibroblast cell line L929 and 
with the human cervix carcinoma cell line ME-180. 
As demonstrated in this example, cyclooxygenase inhibitors, such as 
indomethacin, reduce the toxic effects of high doses of TNF without 
preventing its anti-neoplastic activity. 
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. Therefore, 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. 
The following Table 1 shows the effect of treatment with TNF alone, 
indomethacin alone, ibuprofen alone, or a combination of TNF and either 
indomethacin or ibuprofen, on plasma 13,14-dihydro-15-keto-PGE.sub.2 
("DHK-PG") levels of CD strain male rats. 
TABLE 1 
______________________________________ 
PLASA DHK-PG (13,14-DIHYDRO-15-KETO-PGE.sub.2) 
BEFORE AND AFTER TNF OR SALlNE INTRAVENOUS 
ADMINISTRATION 
Rate Time After TNF or Saline 
Injected With 
0 Time.sup.1 
1 hr 3 hr 
______________________________________ 
[DHK-PG (ng/ml)] 
TNF (Group 1) 
0.40 .+-. 0.05 
4.26 .+-. 0.48 
5.77 .+-. 0.51 
Indomethacin and 
0.42 .+-. 0.07 
0.08 .+-. 0.01 
0.12 .+-. 0.02 
TNF 2 hrs later 
(Group 2) 
Indomethacin and 
0.35 .+-. 0.10 
0.06 .+-. 0.01 
0.12 .+-. 0.03 
Saline 2 hrs later 
(Group 3) 
Ibuprofen and 
0.17 .+-. 0.02 
a.sup.2 0.69 .+-. 0.10 
TNF 2 hrs later 
(Group 4) 
Ibuprofen and 
0.17 .+-. 0.02 
a.sup.2 0.64 .+-. 0.10 
Saline 2 hrs later 
(Group 5) 
______________________________________ 
.sup.1 0 Time measurements were made immediately before administration of 
the TNF or saline. 
.sup.2 a = not determined. 
The following Table 2 shows the effect of treatment with TNF alone, or in 
combination with indomethacin, on the blood pH, pCO.sub.2 and HCO.sub.3 
levels of CD strain male rats. 
TABLE 2 
______________________________________ 
EFFECTS OF TNF AND INDOMETHACIN ON PLASMA 
pH, pCO.sub.2 AND BICARBONATE CONCENTRATlON 
pH pCO.sub.2 (mm Hg) 
HCO.sub.3 (.mu.mol/1) 
______________________________________ 
Saline 7.48 .+-. 0.02 
29.9 .+-. 0.3 
21.4 .+-. 1.1 
(Group A) 
TNF 7.33 .+-. 0.02.sup.1 
22.2 .+-. 2.1.sup.1 
11.1 .+-. 0.9.sup.2 
(Group B) 
Indomethacin 
7.43 .+-. 0.02 
26.0 .+-. 1.6 
16.7 .+-. 1.0.sup.3 
and TNF 
2 hrs. later 
(Group C) 
Indomethacin 
7.35 .+-. 0.06 
39.3 .+-. 5.0 
20.7 .+-. 0.5 
and saline 
2 hrs. later 
(Group D) 
______________________________________ 
.sup.1 p &lt; 0.05 (compared to Group A).? 
.sup.2 p &lt; 0.01 (compared to Group A).? 
.sup.3 p &lt; 0.05 (compared to Group B).?