COMPOSITIONS AND METHODS OF TREATMENT OF A VARIETY OF DISORDERS UTILIZING ETIANATE AND AGONISTS THEREOF

Abstract: A method is presented whereby the side effects but not the activity of biological response modifiers are diminished by the administration of etianate or agonists thereof to a patient receiving with biological response modifiers such as interleukins and tumor necrosis factor.

Description: Description COMPOSITIONS AND METHODS OF TREATMENT OF A VARIETY OF DISORDERS UTILIZING ETIANATE AND AGONISTS THEREOF The research was supported in part by National Cancer Institute Grant No. 5-P30-CA23102, the govern¬ ment may therefore have certain rights to the invention. This is a continuation in part of United States Patent Application Serial Number 07/717,995, filed June 20, 1991 which is a continuation-in-part of United States Patent Application Serial Number 07/673,026, filed March 21, 1991. This invention relates to a method for reducing the side effects of biological response modifiers without reducing their biological activity. Background of the Invention InterleuJin-2 (IL2) , initially described as a paracrine growth factor for T cells, induces non-specific cell-mediated antitu or cytotoxicity against allogeneic and autologous tumor cells when added to unstimulated lymphocytes in vitro. Yron et al., "In vitro Growth of Murine T Cells. V. The Isolation and Growth of Lymphoid Cells Infiltrating Syngeneic Solid Tumors", J. Immunol., 125:238-245 (1981); Do zig et al., "Interleukin-2 Dependence of Natural Killer Cell Activity", J. Immunol., 13:1823- 1841 (1983); and Rosenberg et al., "Biological Activity of Recombinant Human Interleukin-2 Produced in Escherichia coli"- Science, 223:1412-1415 (1984). IL2 has been produced utilizing recombinant DNA techniques. Tanigichi et al., "Structure and Expression of a Cloned cDNA for Human InterleuKin-2", Nature, 302:305-310 (1983). The purified recombinant IL2 has allowed exploration of the IL2 antitumor activity in animals and humans. Kirkman et al., J. Exp. Med. , 162:358 (1985); Cornaby et al. , Transpl. Proc, 20 (Suppl. 1):108 (1988); Rosenberg et al., "A Progress Report on the Treatment of 157 Patients with Advanced Cancer Using Lymphokine - Activated Cells and Interleukin-2 or High-Dose Interleukin-2 Alone", N. Engl. J. Med., 316:889-897 (1987); West et al., "Constant-Infusion Recombinant Interleukin-2 in Adoptive Immunotherapy of Advanced Cancer", N. Engl. J. Med., 316:898-905 (1987); Sosman et al., "Repetitive Weekly Cycles of Recombinant Interleukin-2: Responses of Renal Cell Carcinoma With Acceptable Toxicity", J. Natl. Cancer Inst., 80:60-63 (1988); Fisher et al. , "Metastatic Renal Cell Cancer Treated With Interleukin- 2 and Lymphokine-Activated Killer Cells", Ann. Int. Med., 108:518-523 (1988); and Paciucci et al., J. Clin. One, 7:869 (1989). IL2 administered to animals and humans by constant infusion induces proliferation of natural killer (NK) cells and T lymphocytes. The peripheral blood lymphocytes of patients receiving IL2 acquire the ability to lyse autologous and-allogeneic tumor cells in vitro. A positive relationship exists between IL2 dose- intensity and frequency of response. Paciucci et al., "Immunotherapy with Interleukin-2 by Constant Infusion With and Without Adoptive Cell Transfer and Weekly Doxorubicin", Cancer Treatm. Rev., 16 (Suppl. A):67-81 (1989) . Use of IL2 in cancer treatment is limited by the fact that at effective antitumor doses, the toxic side effects of therapy with IL2 are often severe and require prolonged hospitalization, sometimes in inten¬ sive care units. Rosenberg et al (1987) ; West et al. (1987) ; Sosman et al. (1988) ; and Fisher et al. (1987) . The most frequent IL2 toxicities include obstructive jaundice, renal dysfunction and the capillary leak syndrome, characterized by conspicuous extravascular fluid retention, hypotension and decreased glomerular filtration rate, often leading to cardiovascular and pulmonary compromise. Other significant IL2 toxicities include oral mucositis, nausea, vomiting, protracted anorexia with weight loss, diarrhea, fever, dermatitis and changes in mental status such as depression, confusion and hallucination. Corticosteroids have been used to counteract some of the clinical toxic side-effects of high dose bolus IL2 therapy because of the ability of corticosteroids to suppress undesired inflammatory and immune reac¬ tions. Vetto et al., "Reduction of Toxicity of Interleukin-2 and Lymphokine-Activated Killer Cells in Humans by the Administration of Corticosteroids", J. Clin. Oncol., 5:496-503 (1987). Unfortunately, when the effective abatement by corticosteroids of IL2 toxicities occurred, IL2 antitumor activity was sub- stantially reduced even to extent that IL2 was rendered ineffective. Vetto et al. (1987); and Merchant et al., "Intralesional Infusion of Lymphokine Activated (LAK) Cells and Recombinant Interleukin-2 (rIL-2) for the Treatment of Patients with Malignant Brain Tumors", Neurosurgery, 23:725-732 (1988). Hydrocortisone (17α 21-Dihydroxy-4-pregnane-3,ll, 20-trione) , noted for its broad-scale activities, is utilized widely in medicine for its anti-inflammatory activity. As used herein hydrocortisone also refers to cortisone (also known as hydrocortisol or cortisol) , prednisone, dexamethasone and other similar agonists. Hydrocortisone is the preferred treatment for a variety of conditions such as skin rashes, rheumatoid arthritis, autoimmune diseases (in particular lupus erythematosus) , allergies, thrombocytopenia purpura, he olytic anemia, brain edema, fibrositis and shock. Hydrocortisone has also found great use as a component part of chemotherapy of breast cancer, leukemias and lymphatic cancer and has been found to contain some endogenous anticancer activity. Unfortunately, hydrocortisone causes a number of side effects that limit and sometimes preclude its use. The side effects of hydrocortisone include diabetogenic activity, demineralization of bone leading to fractures and osteoporosis, psychoses, osteonecrosis, increased secretion of gastric acid leading to ulceration of the gastrointestinal tract, obesity, edema and hypoadrena- lism. In addition, hydrocortisone is lymphocytotoxic and therefore immunosuppressive causing exacerbation of infections, development of opportunistic infections and disorders of blood coagulation. Hydrocortisone also promotes etastases of some cancers. Tetrahydrocortisone (3α,17α,21-Trihydroxy- j8-pregnane-ll,20-dione) , the metabolic end product of hydrocortisone, is thought to be devoid of glucocorticoid activity. Crum et al., "A New Class of Steroids Inhibits Angiogenesis in the Presence of Heparin Fragment", Science, 230:1375-1378 (1985). Tetrahydrocortisone, in combination with heparin, has been found to possess anti-angiogenic activity. Crum et al., (1985). Further demonstrating the lack of in vivo activity, tetrahydrocortisone has been found to lack the immunosuppressive effect of hydrocortisone. Langhoff et al. , "The Immunosuppressive Potency in vitro of Physiological and Synthetic Steroids on Lymphocyte Cultures", Int. J. Immunopharmacol. , 9:469-473 (1987). The effectiveness of tetrahydrocortisone in the indications described herein is discussed in United States patent application serial number 07/717,995 which is incorporated herein by reference. Hence, while the following discusses work done with etianate it is expected that similar results would be obtained with tetrahydrocortisone. Etianate, a non-steroidal moiety closely related to steroids, has now been found to possess immunoaug- menting properties comparable to those of tetrahydro¬ cortisone. Etianate is a short chain, 20-carbon 17-5- carboxylic acid end-derivative of steroid metabolism. Monder and Bradlow, "Carboxylic Acid Metabolites of Steroids", J. Steroid Biochem., 8:897-908 (1922). In man, while only 10-22% of urinary metabolites derived from dehydrocortisol are etianic acids, an unmeasured amount may be excreted in the bile. Monder, "Alpha- keto Aldehyde Dehydrogenase, an Enzyme That Catalyzes the Enzymatic Oxidation of Methylglyoxal to Pyruvate", J. Biol. Chem., 242:4603 (1967). Etianic acids are also found in abundance in meconium, an effect of either maternal transplacental transfer or of pregnenolone catabolism. St. Pyrek et al, "Constituents of Human Meconium-I. Identification of 3- hydroxy-etianic acids", J. Steroid Biochem., 18:341-351 (1983), and Zimniak and Lester, "Bile Acid Metabolism in the Perinatal Period", In: Human Gastrointestinal Development. pp. 561-580, ed. Leventhal, Raven Press, New York (1989) . It has also now been found that treatment with etianate was superior to that of hydrocortisone in preventing the occurrence of IL2-induced organ toxicities. Moreover, unlike hydrocortisone, etianate does not adversely effect the IL2-induced generation of lymphokine-activated killer cell mechanisms in vitro and in vivo. Etianate therefore fulfills the requirements for a candidate anti-inflammatory, non- immunosuppressive agent to be used in the clinic in conjunction with biological response modifiers. Summary of the Invention There is disclosed according to the invention a method for ameliorating the side effects of IL2 and other such biological response modifiers without diminishing their activity by administering to patients an effective amount of etianate and agonists thereof concomitant with their administration. A further aspect of the invention is the discovery that etianate may be useful in treating a broad range of physiological disorders commonly treated with hydrocortisone without the adverse side effects often encountered with hydrocortisone. Etianate may also be useful in treating autoimmune disorders and as an adjuvant to vaccines to boost the immune system in response to the antigen(s) present in the vaccine. Brief Description of the Drawings Figure 1 is a set of graphs depicting the genera¬ tion of lytic units against an NK-resistant human melanoma cell line induced by exposure of normal human lymphocytes to IL2, 100 U/ml (I.) in the presence of 0.5 mg/ml of hydrocortisone (F) or etianate (Et) for 24 (panel 1) and for 72 hours (panel 3) . Panel 2 shows results of cultures exposed to steroids for 1 hr. and then washed three times before addition of IL2. Figure 2 is a set of graphs depicting total lytic units j o/mouse recovered after treatment with IL2, IL2 plus hydrocortisone (F) and IL2 plus etianate (Et) against YAC, an NK sensitive cell line and EL4, a syngeneic NK-resistant tumor. Figure 3 is a set of graphs depicting murine alanine aminotransferase (ALT) (panel 1) and bilirubin (panel 2) determined one day after the tenth injection of IL2, IL2 plus hydrocortisone (F) , IL2 plus etianate (Et) or D5W. Detailed Description of the Invention As noted above, the effective use of IL2 in antitumor therapy has been hampered, indeed virtually precluded, by its side effects, e.g. hepatic toxicity. While hydrocortisone has been found to be an effective countermeasure to these side effects, it unfortunately also counters the antitumor activity of IL2 by suppressing lymphokine-activated killer cell activity. It has now been found that etianate lacks the n vitro and in vivo immunosuppressive effects typical of active steroids but retains anti-inflammatory activity. Etianate has now been found to prevent hepatic toxicity induced by administration of IL2 to mammals, but unlike hydrocortisone, etianate does not preempt the i munoaugmenting and lymphoproliferative effects of IL2 in vivo and in vitro. Etianate also prevented IL2-induced hyperbilirubinemia in the mouse and in the rat; in the latter it also protected against thrombocytopenia. IL2 plus etianate did not lead to the severe weight loss seen in animals treated with IL2 plus cortisone. Lymphocytopenia and abrogation of responsiveness to phytohemagglutinin (PHA) in vitro. caused by hydrocortisone, did not occur in animals treated with etianate. Lastly, and perhaps most importantly, natural-killer cell functions and lymphokine-induced tumor cell cytotoxicity, a phenomenon upon which immunotherapy with IL2 is predicated, were preserved by etianate, in contrast to their significant decrease during IL2 plus hydrocortisone treatment. Etianate thus enables the use of IL2 in immunotherapy as a modulator of lymphokine-induced organ toxicities. The molecular mechanisms of systemic and organ toxicity induced by IL2 have not been well defined. In one instance, IL2-induced thrombocytopenia, the toxic side effects are thought to be caused by mononuclear cell release of thromboxane A2 (or A2-like substances) resulting in peripheral platelet destruction. It has recently been found that, subsequent to A2 release, platelets degranulate and release α-2 granules. Factors contained in α-2 granules include proteins with anti-cancer activity such as transforming growth factor β (TGFjS) and platelet factor 4 (PF4) and are found in the plasma of patients within a few hours of IL2 infusion. The ability of etianate to ameliorate thrombocytopenia in IL2 treatment may be due to blockage of the effects of thromboxane A2, α-2 granules, TGFjS and/or PF4 indicating that etianate may be useful in treating side effects encountered in treatment with these factors. It is postulated here without limiting the inven¬ tion, that IL2 toxicity stems from the secondary release of other factors, such as IL1, IL3, IL6, PF4, TGF/3, tumor necrosis factor (TNF) and other like factors recognized to be augmented by IL2. The ability of etianate to ameliorate the side effects of IL2 may be due to its ability to reverse the effects of other factors released in response to IL2 treatment. Thus, etianate treatment may be effective in ameliorating the side effects induced by treatment with other factors including but not limited to interleukins such as IL1, IL3, IL4, IL6, PF4, TGFS, thromboxane A2, α-2 granules, TNF and natural or recombinant preparations of α, β and y interferons. Such factors are herein termed "biological response modifiers". Etianate may act directly on the side effects induced by the factors released in response to IL2 infusion and thus have wide applicability in the amelioration of the side effects induced by a broad range of biological response modifiers. Thus, it is within the scope of the invention to employ etianate to ameliorate the side effects of a broad range of substances which are otherwise known to be beneficial in the treatment of various diseases. In preliminary in vitro studies the effects of hydrocortisone on IL2-induced tumor cytotoxicity were compared to those of several corticosteroid isomers, metabolites or closely related moieties, allegedly biologically "inactive". Having found that inhibition of lymphokine dependent cell-mediated cytotoxicity for tumor cells was maximal for hydrocortisone and minimal for etianate, the activity of etianate in in vitro and in vivo IL2 immunotherapy models was further investigated. The results confirmed prior conclusions that while hydrocortisone diminished to some extent the IL2 side effects, it decreased the effectiveness of IL2. In addition, hydrocortisone, while preventing hyperbilirubinemia, caused extensive hepatic necrosis which was documented both histologically and serologically. On the other hand, it was found that etianate did not seriously alter the effectiveness of IL2 and was shown to be even more effective than hydrocortisone in diminishing certain IL2 side effects. The effects of equimolar concentrations of hydrocortisone were compared to those of etianate on the induction of lymphokine activated killer cells i vitro and it was found that cortisone decreased the effectiveness of IL2 whereas etianate did not affect IL2 activity. The antitumor effects of etianate were compared to those of hydrocortisone. It was found that etianate had an antitumor effect on certain tumors as did hydrocortisone. Higher concentrations of etianate were required to achieve an affect comparable to that of hydrocortisone. It is thus an aspect of the invention to use etianate alone as an antitumor agent. The effects of equimolar concentrations of hydrocortisone and of etianic acid on the induction of lymphokine activated killer cells in vitro was studied and it was found that hydrocortisone but not etianic acid inhibited the generation of unrestricted tumor cytotoxicity. _£n vivo administration of etianic acid to mice receiving IL2 did not inhibit the generation of cytotoxic cells against two syngeneic tumor cell lines whereas hydrocortisone did cause inhibition. The examples presented below utilize etianic acid, however, other etianates as defined herein, are equally effective and are encompassed by the present invention. The invention further encompasses agonists of etianate. Agonists are compounds that mimic at least some of the effects of compounds by interaction with the appro¬ priate physiological receptor. Etianate also protected against IL2-induced thrombocytopenia, another immunologically mediated effect of IL2. Paciucci et al. , "Thrombocytopenia during Immunotherapy with IL2 by Constant Infusion", Am. J. Med., 89:308-312 (1990). Glucocorticoids did not reverse thrombocytopenia caused by IL2, reversed IL2-induced lymphocytosis (causing instead severe lymphocytopenia) and prevented eosinophilia. Etianate, at equimolar doses did not have any effect on lymphocyte or eosinophil counts. Furthermore, the data presented below indicate that etianate may possess previously undetected immuno- logical activity. Etianate may enhance, or at very high doses may depress, immune responses. At the doses described below in the rat model, etianate appeared to decrease the self reactive cellular reaction induced by IL2 in the liver. Etianate may thus possess broader and therapeutic effects on the cell-mediated disreac- tivity typical of autoimmune diseases as well as other conditions caused by an abnormal activation of the immune system. Etianate may thus be useful in a method of treating autoimmune diseases. Etianate can be administered to a patient by any method known in the art provided that pharmaceutically effective levels are reached. The effective amount and method of administration of the particular etianate formulation may vary based on the nature of the condition and disorder being treated, its severity, the age of the patient and other factors evident to one skilled in the art. In general, etianate compounds are pharmaceutically acceptable due to their low toxicity in the therapeutic dosage range, stability and ability to be incorporated into a wide variety of vehicles for numerous routes of administration. Etianate can also be used in treating humans suffering from adverse health conditions which normally respond favorably to treatment with hydrocortisone. A list of such conditions is provided above and composi¬ tions containing etianate suitable for treating such conditions are provided below. Etianate may be useful in treating autoimmune diseases particularly those involving collagen. Such autoimmune diseases include but are not limited to systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis and scleroderma. Methods of administration of etianate include any of those described herein. Etianate may also be useful as an adjuvant to vaccines to boost the immune response to the vaccine. Methods of vaccine production and administration are well known in the art and will not be described in detail herein. Etianate may be administered as part of the vaccine composition or just prior to or after vaccination in a separate treatment. According to the present invention, in order to obtain the beneficial effects of etianate, pharmaceutically acceptable etianate-containing compounds are administered to the patient in an amount sufficient to provide therapeutic levels of etianate. Etianate can be administered in a variety of ways. The route(s) of administration useful in a particular application are apparent to one of skill in the art. Routes of administration include but are not limited to topical, transdermal, parenteral, gastrointestinal, transbronchial and transalveolar. It is preferred that etianate be administered by constant infusion when etianate is administered in conjunction with a biological response modifier. Such infusion can be accomplished in a variety of ways for instance by a subcutaneous pump or osmotic pump or by intravenous administration. Formulations of etianate suitable to be applied topically include but are not limited to implants, ointments, creams, rinses, gels, sterile solutions and liposomally encapsulated vesicles. Formulations suitable for transdermal administration include but are not limited to suspensions, oils, creams and ointments applied directly or attached to a protective carrier such as a patch. Formulations suitable for parenteral administration include but are not limited -to sterile solutions for intravenous, intramuscular, or subcutaneous injection or infusion, including infusion pumps. Formulations suitable for gastrointestinal administration include, but are not limited to, pills or liquids for ingesting and suppositories for rectal administration. Formulations suitable for transbronchial and transalveolar administration include, but are not limited to, various types of aerosols for inhalation. The above-mentioned formulations are meant to describe but not limit the methods of administering etianate. The methods of making the various formulations are within the ability of one skilled in the art and will not be described in detail here. Etianate is useful for the treatment of conditions previously treated with hydrocortisone and in the manner treated with hydrocortisone. In general, due to the decreased glucocorticoid activity and decreased side effects of etianate compared to hydrocortisone, etianate can be administered in dosages exceeding that of hydrocortisone. Generally, in the case of human patients, etianate is administered in the range from a level found to yield diminished symptoms up to a level just below that which is toxic. Methods of determining toxicity are known in the art and will not be described in detail here. Holland, "Clinical Pharmacology in Patients With Cancer", Int'l. Encyc. of Pharmacol., Sect. 6, 11:597-616 (1966) Pergamnon Press, Ltd. New York. When administered systemically, etianate can be administered in a range of from about 1 mg to about 10 g per day, more preferably about lb mg to about 1 g per day, and even more preferably about 100 mg to about 500 mg per day. Typically in the case of topical administration for rashes and the like, an effective level of etianate would be about 1% in a physiologically acceptable carrier. Administration of etianate compounds would cease once healing has occurred. According to the invention, when etianate is used to ameliorate the side effects of biological response modifiers, etianate may be administered continuously, for instance by constant infusion by a pump or intravenously or by oral consumption of more than one dose per day, for example in multiple doses around the clock. Such dosages can be for instance, four times daily or every six hours. It is contemplated that the biological response modifiers and etianate be administered in combination. As used herein, in combination means either a single mixed dosage (e.g., a single injectable solution) or separate compositions of biological response modifiers and etianate respectively, administered during the same time period, or closely related or overlapping time periods. For example, the treatment composition according to the present invention could include biological response modifiers and etianate in separate vials suitable for injection, or IL2 in injectable form and etianate in orally administrable (e.g. tablet) form. The following examples are meant to illustrate but not limit the present invention. Example 1 The Effect of in vitro Administration of Hydrocortisone and Etianate to Lymphocytes Treated with IL2 Lymphocytes were gradient separated from hepar- inized peripheral blood of healthy volunteers and cultured 2xl0 6 /ml in RPMI 1640 medium (Gibco, Grand Island Biologic Co.) supplemented with 20% fetal bovine serum. Exogenous recombinant IL2 (Cetus Corporation, Emeryville CA) , was added at 600 IU/ml. Hydrocortisone 0.05 mg/ml (Solucortef, Upjohn Co., Kalamazoo, MI), etianate (Sigma compound designated T7905) 0.25 mg/ml were diluted in 5% dextrose in water (D5W) . IL2- induced unrestricted tumor cytotoxicity was assessed using standard 8-hour 51 Chromium release assay against DND-1A, a melanoma cell line developed in our labora¬ tories. Percent tumor lysis at different effector/ target cell ratios was converted into lytic Units 20% (LU 2 o) defined as the number of effector cells contained in each culture that could lyse 20% of target tumor cells. The results presented in Figure 1 show generation of lytic units against an NK-resistant human melanoma cell line induced by exposure of normal human lympho¬ cytes to IL2, 100 U/ml (I) in the presence of 0.05 mg/ml of hydrocortisone or etianate for 24 (panel 1) and for 72 hours (panel 3) . Figure 1, panel 2 shows results of cultures exposed to the compounds for 1 hour and then washed three times before addition of IL2. In panel 2C, cultures were pretreated with etianate for one hour and then with hydrocortisone before the addition of IL2. Exposure to hydrocortisone con¬ tinuously or for only 1 hour before the addition of IL2 decreased the generation of lytic units against (p < 0.05) compared to IL2 only. There was no effect in cultures exposed to etianate. Exposure to hydrocortisone for 24 hours was not significantly different from one hour only (p > 0.05). Pretreatment with etianate and then hydrocortisone partially, but significantly, abrogated the inhibitory effects of hydrocortisone. As shown in Figure 1, hydrocortisone but not etianate inhibited the generation of unrestricted tumor cytotoxicity. Substantial inhibition of IL2 activity was also induced by exposure to hydrocortisone for one hour before stimulation with IL2. The partial protection against hydrocortisone-induced inhibition of lymphokine-activated tumor cytotoxicity seen when lymphocytes were preincubated for one hour with etianate may relate to the immunoenhancing effects of the drug since it is believed that the two compounds do not use the same receptor. Example 2 The Effect Of in vivo Administration Of Hydrocortisone And Etianate In C57BL/6 Mice Treated With IL2 The immunologic effects of in vivo administration of IL2 were quantified by determining, in each animal, the cytotoxicity of spleen cells against YAC, an allogeneic, A/J-derived NK-sensitive cell line and EL4, a syngeneic NK-resistant T-cell leukemia/lymphoma line. Total lytic units 20/mouse were recovered after treatment with IL2, IL2 plus hydrocortisone and IL2 plus etianate against YAC, and EL4. Six to 12 week-old male C57BL/6 mice (Jackson Laboratories, Bar Harbor, Me.) were randomized into control and treatment groups of 12 to 18 mice per experiment. The treated group was injected intraperi- toneally with IL2, 10 5 units in 0.2 ml of D5W daily for five days and the control with 0.2 ml of D5W only. After a 2 day interval, each group was randomized into 3 subgroups of 4 to 6 animals each. In the IL2 pretreated group, mice in each subgroup were injected daily for 5 days, from day 8 to day 12, with IL2, IL2 plus cortisone and IL2 plus etianate. Equimolar doses of both drugs, 50 mg/kg, were administered diluted in 0.2 ml of D5W. Control mice received a second 5 day-treatment with D5W intraperitoneally (i.p.) As shown in Figure 2A, hydrocortisone but not etianate, significantly decreased resident cytotoxic mechanisms against the NK-sensitive YAC tumor cell line. In mice treated with IL2 there was augmentation of lysis against this target as well as generation of cytotoxicity against the NK-resistant EL4 leukemia. Administration of hydrocortisone sharply decreased the generation of total lytic units against YAC (Student's T test: p < 0.0001) and EL4 (p = 0.008), whereas etianate did not suppress IL2 dependent tumor cyto- toxicity. The difference between hydrocortisone and etianate was significant (p = 0.006) for YAC and p = 0.008 for EL4) . Hydrocortisone significantly suppressed the generation of anti-EL4 cytotoxicity also at one tenth of the dose, 5 mg/kg while etianate, even at 500 mg/kg, ten times the dose shown in Figure 2, produced insignificant inhibition of cytotoxicity for EL4 (p = 0.977). Example 3 Effect of Hydrocortisone and Etianate on IL2 Toxicity in vivo Analysis of individual mouse serum samples, showed that both hydrocortisone (p = 0.037) and etianate (p « 0.014) abrogated IL2-induced hepatic toxicity, measured by the elevation of alanine aminotransferase (Figure 3A) and serum bilirubin (Figure 3B) . Signifi¬ cant nephrotoxicity from IL2 was not encountered. Mice were euthanized by cervical dislocation. Individual mouse serum samples were used for the determination of serum chemistries. Murine alanine aminotransferase (ALT) (panel 1, Figure 3) and bili¬ rubin (panel 2, Figure 3) were determined one day after the tenth injection of IL2, IL2 plus hydrocortisone, IL2 plus etianate or D5W. Bilirubin, determined with standard colorimetric techniques, is not reported for those samples in which hemolysis occurred. There was IL2-induced increase of serum bilirubin and ALT in mice receiving IL2 only. Significant abatement of IL2- induced hepatic toxicity was seen in mice treated with hydrocortisone (p - 0.047) or with etianate (p - 0.014). No differences in the four treatment groups were seen in the serum alkaline phosphatase, glutamic-oxalacetic transaminase, blood urea nitrogen and creatinine. Panel C shows the serum bilirubin levels obtained in male Sprague-Dawley rats after 5 days of treatment with IL2 and the compounds by constant infusion using osmotic infusion pumps according to the manufacturer's instructions (Alzet model 2ML1, Alza, Palo Alto, CA) . These pumps have an internal reservoir of 2 ml of which 1.7 ml are delivered over a 7-day period with a mean pumping rate of 10 ml/hr. Pumps were implanted subcutaneously 2 to 3 cm below the scapulae and were removed on day 8 of treatment under identical anesthesia. Cohorts of 2-3 rats in each experiment received control diluent (D5W) , recombinant human IL2 10 6 U/day, IL2 in combination with hydrocortisone or etianate both at 40 mg/kg/day. There was no substantial difference amongst the four treat¬ ment groups in other serum chemistries. Further experiments were performed in a rat model, in which subcutaneous infusion pumps (for the adminis¬ tration of IL2, corticosteroids and control diluent) and indwelling central catheters (for frequent blood sampling during treatment) allowed determination of the kinetics of IL2 toxicity and of the effects of the compounds. In this model, hepatic dysfunction was also a prominent effect of treatment with IL2 (Figure 3C) . Peak elevation of serum bilirubin was observed on day 5 of treatment in 7 of 7 rats receiving IL2 only (mean bilirubin 4.3 mg/dl, median 4.7) in contrast to 7 controls which had no measurable bilirubin levels (p < .009). The cohorts of rats receiving IL2 plus hydrocortisone or etianate had significantly lower serum bilirubin levels than those receiving IL2 only (0.7 mg/dl and 1.4 mg/dl respectively, p = 0.014 for both) . Peak elevation of the alanine aminotransferase (ALT) was also seen on day 5 of treatment in all animals treated with IL2 (Table 1) . Table 1 shows the toxicity grading of platelets, bilirubin and alanine aminotransferase (ALT) in 7 rats treated with IL2, IL2 plus hydrocortisone (F) , IL2 plus etianate (Et) or 5% dextrose (CTR) by constant subcu¬ taneous infusion for 7 days. The grading was carried out according to standard World Health Organization Criteria used for human Phase I studies on deter¬ minations done at baseline and every 2-3 days during treatment. During treatment there was a substantial increase of serum bilirubin in the IL2 only treatment group compared to untreated controls (p = 0.003) which peaked on day 5 (see Figure 3, panel C) . Concomitant administration of hydrocortisone (p = 0.003) and etianate (p = 0.031) abrogated the toxicity in 6 of 7 rats each. Thrombocytopenia was worse in rats treated with IL2 only (p = 0.076); treatment with hydrocortisone (p = 0.322) and etianate (p = 0.036) decreased the median toxicity grade. There was no detectable IL2-induced change in renal function; all treatment groups, inclu¬ ding controls, had abnormal alkaline phosphatase and aspartic aminotransferase. For repeated blood sampl¬ ing, a polyethylene catheter was inserted into the superior vena cava via the left external jugular vein. To maintain patency, catheters were flushed daily with 0.5 ml of preservative-free heparin, 250 units/ml. The results shown in Table l indicate that ALT was marginally higher for animals receiving IL2 plus hydrocortisone, whereas no difference was discernible between the IL2 only and IL2 plus etianate groups. Elevations of alkaline phosphatases and of the aspartic transaminases occurred in all animals regardless of treatment. In order to elucidate IL2-induced hepatic dysfunction, livers of rats who died during treatment, following blood sampling or as a consequence of surgical complications when repositioning indwelling catheters were examined histologically. The liver preparations were stained with hematoxylin-eosin. Histological examination showed extensive, periportal mononuclear cell infiltrates with diffuse parenchymal infiltration in animals receiving IL2 only. In those treated with IL2 plus hydrocortisone there was uniform, extensive hepatocyte vacuolization without interstitial infiltrates and in those treated with IL2 plus etianate there were minimal infiltrates in the periportal spaces. The finding of hyperbilirubinemia with extensive periportal and intraparenchymal mono¬ nuclear cell infiltrates induced by treatment with IL2 in rats is similar to the IL2-induced obstructive jaundice experienced by cancer patients receiving IL2 by constant infusion. Thrombocytopenia, a common effect of IL2 in humans when the lymphokine is administered by constant infu¬ sion, was also seen in rats (Table 1) . Paciucci et al. (1990) . The median increase of toxicity grade from baseline to nadir was borderline significantly higher in the IL2 only treatment group than in the controls (control median = 1, IL2 median = 2.5, p = 0.076). Treatment with hydrocortisone and etianate decreased the median increase in toxicity grade from baseline to nadir compared to IL2 only (median IL2 + hydrocortisone = 0.5, median IL2 = etianate = 0.5, p = 0.060). Unlike what is commonly observed in humans, clear¬ ing of lymphocytes from the peripheral blood does not occur in rats during administration of IL2 by constant infusion. Absolute lymphocytopenia, however, occurred in the group of animals receiving IL2 plus hydrocorti- sone in whom the average baseline lymphocyte count was 7510/μl and decreased after 7 days of treatment to 2445/μl (p < .0001 by Student's t test). No substan¬ tial change from baseline lymphocyte counts was seen in the three other treatment groups and thus lymphocyto- penia was attributed to the lymphocytotoxic effects of glucocorticoids. An additional corroboration of immunosuppression induced by hydrocortisone is the finding of an 82% decrease of in vitro proliferative responses to phytohemagglutinin (PHA) in this treatment cohort (p = 0.015 by Student's t test) whereas there was no substantial change in the m vitro proliferation to PHA in the rats receiving D5W, IL2 only or IL2 plus etianate. Unlike what is commonly experienced by humans receiving immunotherapy with IL2, there was no evidence of fluid retention in rats, who indeed lost weight during treatment with IL2. Weight loss was worse in animals receiving IL2 plus hydrocortisone, in whom the average weight after treatment was 81% of baseline (p = 0.003). In contrast, on day 8 of treatment, the average weight of control rats was 101% of baseline and that of rats receiving IL2 only and IL2 plus etianate was 93% (not significant) . The difference between hydrocortisone and etianate was significant (p = 0.032) . It is clear by the foregoing examples the etianate, and not hydrocortisone, abrogates the toxic side effects of IL2 without diminishing IL2 antitumor activity. Example 4 Materials and Methods In view of the results obtained in Examples 1-3, the following experiments described in Examples 5-11 were performed. In the following examples, the mate- rials and methods described below were used to obtain the data presented in Tables 2-8 and the histological data discussed in the Examples. Lymphocyte cultures: Peripheral blood mononuclear cells were obtained from healthy volunteers by gradient sedimentation with Ficoll-Hypaque. For culture, cells were suspended in RPMI 1640 medium (Gibco Laboratories, Grand Island, NY) with 10% heat inactivated fetal bovine serum (Gibco) supplemented with penicillin/streptomycin. Steroids: Hydrocortisone sodium hemisuccinate was purchased from the UpJohn Company, Kalamazoo, MI. Etianic acid, compound T7905, was purchased from Sigma (Saint Louis, MO) . Both compounds were diluted with 5% dextrose in water (D5W) . Short-Term Activation with Interleukin-2: Recombinant human IL-2, generously provided by the Chiron Corporation, Emeryville, CA, was reconstituted in D5W. Ten x 10 6 lymphocytes in 10 ml medium were activated with IL2, 600 IU/ml in 25 cm 2 upright culture flasks (Corning Glass Works, Corning, NY) . Cultures were incubated for 24 hours at 37°C in a humidified atmosphere of 5% C0 2 . In each experiment, unstimulated, control lymphocytes were cultured under the same conditions. In cultures in which drug exposure was constant, hydrocortisone or etianate, 0.05 - 0.25 mg/ml, were added immediately after IL2. To determine the effect of short-term exposure to steroids, cell aliquots were also exposed to steroids for one hour. After washing three times, cell concentrations were adjusted for culture and IL2 was added. Parallel, control-stimulated cultures were exposed to mock- steroid, incubated and washed three times prior to the addition of IL2. Mixed Lymphocyte Cultures: Five x 10 6 responder cells were cultured for 7 days in 5 ml medium with 10% heat inactivated pooled human serum and stimulated with an identical amount of allogeneic lymphocytes irradiated with 4000 Rads (at a rate of 400 Rads/min) . Untreated and unstimulated allogeneic cells were also cultured as a source of allospecific target cells under similar conditions. Allosensitized cultures were treated with steroids as described above. To determine in vitro proliferation induced by allosensitization and steroid treatment, upon termination of cultures, 0.1 ml aliquots of each were labeled with 0.2 μCi 3 H-thymidine (New England Nuclear) for 8 hours. Labeled cultures were harvested (Skatron) on nylon fiber filters and incorporation of radioactivity was evaluated by liquid scintillation in a Packard Tricarb model 1900-CA beta counter. Cytotoxicity: After harvesting, cells were washed, suspended at 2 x 10 6 cells/ml and 0.1 ml aliquots dispensed in round-bottom 96-well microtiter plates together with 0.1 ml containing 5 x 10 3 target cells that had been labeled with 100 μCi of 51 Cr (New England Nuclear, MA) . In each experiment, 3 effector/target cell ratios were studied (40:1, 10:1 and 2.5:1) in 4 replicate wells. Microcytotoxicity trays were incubated at 37°C in a humidified atmosphere of 5% C0 2 and after 8 hours radioactive supernatants were collected with Skatron harvesting frames (Skatron Inc. , Sterling, VA) and percent lysis determined according to the formula: % lysis = (cpm test - cpm spontaneous release) x 100 (cpm maximum release - cpm spont. release) where spontaneous release is the radioactivity of 6 replicate wells in which 0.1 ml IN HCl was added to 0.1 ml of labeled target cells and spontaneous release that of wells in which target cells only were cultured in 0.2 ml medium without effector cells. Specific percent lysis was converted into lytic units 20% (LU 20 ) , defined as the number of effector cells contained in each culture sample that could lyse 20% of 5 x 10 3 target cells. Immunophenotvping: Monoclonal antibodies (mAb) Leu4 (CD3), Leul2 (CD19) and anti-IL2 receptor (CD25) , conjugated with either fluorescein or phycoerythrin were obtained from Becton-Dickinson. Aliquots (150 μl) of cultured effector cells at 10 6 /ml were conjugated with each mAb (final concentration 2 μg/ml) for 30 minutes at room temperature. Cells were washed twice with phosphate buffered saline and fixed with 1% para- formaldehyde. Cell subpopulations were defined by the backgating procedure using the simultest Leuko-Gate staining technique and Simultest software according to the manufacturer's instructions (Becton-Dickinson) . Cells*were analyzed for differential staining patterns. Two parameter contour maps were integrated to determine the percentage of positive and negative cells. The analysis was carried out on a FAC Star II cell-sorter with four decades of amplification. Animal Studies: Six to 12 week-old male C57BL/6 or BALB/c mice (Jackson Laboratories, Bar Harbor, ME) were used to study the effects of steroids on the in vivo generation of lymphokine-induced tumor killer cells with IL2 and steroids. Mice were inoculated subcutaneously with IL2, 6 x 10 5 IU in 0.1 ml D5W daily for 5 days/week for 2 consecutive weeks. Cohorts of 4 to 10 animals in each experiment also received bolus hydrocortisone or etianate, 60 mg/kg. At the end of treatment animals were euthanized by cervical disloca- tion and spleen cells were used as effector cells against radiolabeled tumor target cells. Blood, ob¬ tained from the inferior vena cava with 25 gauge needles, was clotted and the serum was separated by centrifugation. A standard panel of serum chemistries were obtained according to standard procedures to assess renal function (blood urea nitrogen -BUN- and creatine) and hepatic function (total bilirubin, alkaline phosphatases, gluta ic-pyruvic and glutamic- oxalacetic transaminases) . In vivo Allogeneic Sensitization: BALB/c mice were immunized twice in an 8 day period with 40 x 10 6 C57BL/6 spleen cells. Three days after the last inoculation of allogeneic cells, cohorts of mice were randomized to receive daily subcutaneous injections of control diluent (D5W) , IL2 only, or IL2 plus steroids as described above. Murine Tumor Target Cells: To determine the effects of treatment with IL2 and IL2 plus steroids on the generation of cell-mediated antitumor responses in mice, two C57BL/6 tumors were used as a source for target cells: EL4, a benzpyrene induced T cell leukemia/lymphoma line and P20, a non-metastatic variant of B16 melanoma. Effects on natural killer (NK) cytotoxicity were evaluated against YAC, a NK- sensitive lymphoma. In vivo effects of IL2 and Steroids in rats: confirmatory experiments to further characterize the protective effects of steroids were carried out in a rat model. Sprague-Dawley male rats, each weighing 250-300 grams, were anesthetized with USA xylazine (Rompun, Cutter Laboratories, Shawnee, KS) , 12 mg/kg intramuscularly and ketamine (Ketalar, Parke-Davis, Morris Plains, NH) , 30 mg/kg mtraperitoneally prior to the placement of subcutaneous osmotic pumps (Alzet model 2 ML1) with a mean pumping rate of 10 μg/hr over a 7 day period. Pumps were implanted through a small incision made in the skin 2 to 3 cm below the scapulae and were inserted with the flow moderator pointing away from the incision. The skin incision was closed with sutures or wound clips. One 2 ml sample of blood specimens were obtained at baseline and after 5 and 7 days of treatment to determine serum chemistries and complete blood counts during treatment with IL2. In each experiment cohorts of three rats were treated with constant infusion of control diluent (D5W) , IL2, 29 x 10 6 IU/kg/day, IL2 in combination with constant infusion hydrocortisone, or etianate, 40 mg/kg/day each. Example 5 Effects of Hydrocortisone and Etianic Acid on IL2-induced Cytotoxicity and IL2-receptor Expression in Human Lymphocytes In an experiment to assess the effects of hydro- cortisone and etianate on the cytotoxicity of IL2- induced cells it was found that etianate did not suppress the i vitro generation of anti-tumor cytotoxicity in cultures of normal human lymphocytes and conferred partial protection to subsequent exposure to hydrocortisone. Inhibition of cytotoxicity by hydrocortisone was associated with upregulation of the IL2 receptor, a finding already reported in different systems. Fernandez-Ruiz et al., "IL2 Protects T-cell Hybrids from the Cytolytic Effect of Glucocorticoids. Synergistic effect of IL2 and Dexamethasone in the Induction of High Affinity IL2 Receptors", J. Immunol., 143:4146-4151 (1989). The data depicted in Table 2 were obtained as follows. Effector cells (b) and (c) were cultured for 24 hours in the presence of hydrocortisone or a four¬ fold excess of etianate before the addition of IL2, 600 u/ml; all effectors underwent mock exposure to steroids (like effectors (c)-(f), below) and were washed three times before addition of exogenous IL2 or steroids. Exposure to IL2 or steroids was for 24 hours. Effector cells (d) and (e) were washed three times after expo- sure to the respective steroids for one hour. Effector cells (f) were exposed for one hour to etianate and subsequently for one hour to hydrocortisone, washed three times and exposed to IL2. After a 24 hour culture, IL2-induced activity was tested against a melanoma cell line. The results showed that hydrocor¬ tisone, but not etianate, greatly decreased lytic activity, regardless of duration of treatment. Pretreatment of effector cells with etianate and then exposure to hydrocortisone conferred protection against the hydrocortisone-related suppression of IL2-induced cytotoxicity (p = 0.014 by Wilcoxon Signed Rank Test). As shown in Table 2, when normal human lymphocytes were activated with IL2 for 24 hours, there was a five- fold decrease of the total number of lytic units recovered from cultures treated with hydrocortisone. A significant inhibition (p = 0.014) of lymphokine- activated killer cell activity was also observed when lymphocytes were exposed to the same dose of hydrocor- tisone, 0.05 mg/ml for only one hour. In contrast, etianate slightly increased the activity of IL2 (p = 0.025) and pre-treatment with etianate and then exposure for one hour to hydrocortisone before addition of IL2 conferred partial protection against the inhibi¬ tory effects of hydrocortisone on the generation of tumor cytotoxicity (p = 0.014). The unlikely possibility that this effect was consequent to competitive receptor binding of the two steroids was ruled out by receptor binding experiments with tritiated hydrocortisone. Since the cell viability observed in steroid-treated cultures was similar to that of control stimulated cells, the inhibitory effects of hydrocortisone and their relation to selective alterations in the frequency of effector cell subpopulations was studied. As shown in Table 2, surface marker studies indicated that activation with IL2 and hydrocortisone induced a moderate, but significant, decrease in the frequency of CD3 + cells and, at the same time, upregu- lated the expression of the high-affinity IL2 receptor (CD25) on both CD3 + and CD3" cells. Changes on NK cell subsets were not seen. These results suggest that the adverse effects of hydrocortisone on cell-mediated tumor cytotoxicity are not, at least in the m vitro system, consequent to lymphocytotoxicity. The finding that, in vitro. etianic acid antagonizes the immunosuppressive effects of hydrocortisone raises the possibility that other untoward effects of steroids may be blocked by etianate. Example 6 Effect of Hydrocortisone and Etianate on the Generation of Allospecific and MHC-unrestricted Cytotoxicity Following in vitro Allosensitization Lymphocyte allosensitization generates effector cells that are cytotoxic not only for allospecific target cells but also for allogeneic and syngeneic tu or cells. This is a phenomenon associated with autocrine release of IL2 during allosensitization. Paciucci et al. , "Lysis of Syngeneic Solid Tumor Cells by Alloantigen Stimulated Mouse Cells", J. Immunol., 124:370-377 (1980a); Paciucci et al., "Requirements of T cells in Alloantigen-Induced Generation of non-T cell Mediated Cytotoxicity Against Syngeneic Mouse Sarcoma Cells", J. Immunol., 125:36-38 (1980b); and Macphail et al., "Phenotypic Heterogeneity of Anti-Syngeneic Tumor Killer Cells (ASTK) Generated in Allogeneic Mixed Lymphocyte Reactions", J. Immunol., 132:3205-3212 (1984) . It has previously been reported that in vitro allosensitization induces not only generation of allospecific cytotoxic effector T cells but also generates non-specific cytotoxicity against allogeneic and syngeneic tumor cell lines n vitro. Yron et al. (1981) ; Domzig et al. , "Interleukin-2 Dependence of Natural Killer Activity", J. Immunol., 13:1823-1841 (1983); Rosenberg et al. (1984); and Taniguchi et al. (1983) . The following experiment was designed to test the effects of hydrocortisone and etianate on this phenomenon. The results shown in Table 3 were obtained as follows. Peripheral blood lymphocytes from healthy volunteers were allosensitized for 7 days at 37°C in a humidified atmosphere of 5% C0 2 in RPMI 1640 medium supplemented with 10% heat-inactivated human serum. Stimulating cells were equal numbers of irradiated (4000R over 2.5 minutes) allogeneic lymphocytes or autologous lymphocytes (controls) . Cultures were added with hydrocortisone or etianate (0.05 and 0.2 mg/ml each, respectively) . After culturing, the cells were harvested, washed, counted and used as effectors against 51 Cr labeled allospecific normal target cells and an unrelated melanoma cell line. Cytotoxicity assays were performed at different effector to target ratios. Simultaneously, parallel cytotoxicity trays were set up with quadruplicate ratios (100:1, 20:1 and 5:1) of effector and target cell mixtures. These were incubated in the presence of exogenous IL2, 1000 units/ml. After 8 hours, super¬ natants were harvested and cytotoxicity was determined according to the formula: % lysis = (cpm test - cpm spontaneous release) x 100 (cpm maximum release - cpm spont. release) where spontaneous release is the radioactivity of wells to which no effector cells were added and maximum release is the radioactivity of wells in which maximum lysis was obtained by addition of IN HCl. Cytotoxicity results were then expressed as lytic units 20 , in which a lytic unit is the number of effector cells, for each effector cell population, capable of lysing 20% of target cells. p = Students t test As the data in Table 3 indicate, incubation of mixed leukocyte cultures with hydrocortisone ablated the generation of both allospecific and non-MHC res¬ tricted cytotoxicity. Allospecific lysis mediated by cells sensitized in the presence of etianate 0.05 mg/ml was substantially higher than that mediated by control sensitized effector cells after short-term incubation with or without IL2, 500 u/ml. Etianate decreased, however, the lytic ability of effector cells against unrelated tumor cells. Whereas short-term incubation with IL2 after allosensitization augmented the tumori- cidal activity of control-generated effector cells, in the presence of etianate there was a dose-related decrease in the ability of effector cells to mediate non-MHC-restricted cytotoxicity and less lytic units against tumor cells were recovered after short-term exposure to IL2. Table 3 further shows that hydrocortisone also abrogated proliferative responses of human lymphocytes in mixed leukocyte cultures (MLC) as well as the generation of cytotoxic effector cells against allo¬ specific target cells. In these MLCs there was also complete inhibition of the MLC-induced generation of MHC-unrestricted responses against tumor cells, a phenomenon that occurs during m vitro allosensitiza¬ tion. Paciucci et al. (1980a); Paciucci et al. (1980b); and Macphail et al. (1984). In etianate-treated MLC there was a modest but significant (p = 0.001) dose-dependent inhibition of cell proliferation but the generation of cytotoxicity against allospecific normal target cells was not affected at either dose used. There was, however, inhibition of unrestricted tumor cytotoxicity at a concentration of 0.2 mg/ml (p = 0.037) which was not restored to normal levels by post-sensitization exposure to IL2. The anti-tumor lytic units decreased 3-fold for etianate at 0.05 mg/ml (p = 0.037) and 6- fold for etianate at 0.2 mg/ml (p = 0.014). Allogeneic stimulation in the presence of hydro¬ cortisone ablated the generation of both types of cytotoxicity. Etianate, while exerting modest inhibi¬ tory effects on the MLC-induced tumor cytotoxicity, did not interfere with allospecific, T cell mediated cyto¬ toxicity and subsequent exposure of allosensitized effector cells to IL2 augmented cytotoxicity against both allospecific and tumor target cells. Example 7 In vivo Effects of Steroids on the Generation of Tumor Cytotoxicity Against Syngeneic and Allogeneic Tumors Following IL2 and on the Generation of Allospecific Cytotoxic Responses Following Alloimmunization The effects of hydrocortisone and etianate on IL2- induced anti-tumor cytotoxicity in vivo were studied. The results obtained are shown in Table 4 and were obtained by the following method. Six to 12 week old male C57BL/6 mice were treated with IL2, 6 x 10 5 IU/day for 5 days/week for 2 weeks. Cohorts of 4 to 10 animals in each experiment were also concomitantly treated with hydrocortisone or etianate, 1.5 mg/day subcutaneously. On the last day of the experiment mice were euthanized and spleen cells were used as effector cells against 51 Cr-labeled tumor target cells, as indicated, to assess the effects of IL2 and steroid treatment. The data indicate that hydrocortisone but not etianate, induced substantial inhibition of IL2- induced tumor cytotoxicity. The data presented in Table 4 indicate that in C57BL/6 mice treated with IL2, 10 5 units five times daily for two weeks, treatment with hydrocortisone prevented the generation of cytotoxicity against the syngeneic P20 melanoma and EL4 leukemia target cells as well as against the NK-sensitive allogeneic YAC cells. Administration of etianate together with IL2 did not impair the generation of lymphokine-dependent tumor cytotoxicity. Similar effects for hydrocortisone and etianate were seen in mice treated with doses ten-fold higher and lower. In mice treated with IL2 there was a higher recovery of splenocytes than in controls treated with mock-IL2 (126 and 145 x 10 6 , respectively) . Significantly higher recovery was always obtained in those mice treated with IL2 plus etianate (161 x 10 6 , p = 0.05) while the opposite was true for those treated with IL2 plus hydrocortisone (105 x 10 6 , p = 0.02). The difference of splenocyte recovery induced by treatment with etianate or hydrocortisone was highly significant (p = 0.005) . Further, the dose-dependent inhibition of unrestricted cytotoxicity during culture with etianate may be viewed as a remnant steroid effect, but this was quite selective and was not seen against allospecific target cells in vitro and m vivo in alloimmune mice. The possibility that this steroidal effect of etianate is consequent to an interference with IL2 production during allosensitization is being investigated. This interference is a mechanism proposed for steroid- induced immunosuppression. Redondo et. al., "Inhibition of Interleukin-2-induced Proliferation of Cloned Murine T-cells by Glucocorticords Possible Involvement of an Inhibitory Protein", Eur. J. Immunol., 18:1555-1559 (1988). Example 8 Effect of Etianate on Allospecific Lysis of EL4 by Spleen Cells of BALB/c Mice Immunized Against C57BL/6 To determine the in vivo effects of steroids on the induction of allospecific responses, BALB/c mice, immunized with C57BL/6 spleen cells, were treated with D5W or with etianate, 60 mg/kg/day. Two additional groups also received IL2 or IL2 plus etianate. The data in Table 5 were obtained by the following method. Male BALB/c mice, 6 to 12 weeks old, were immunized with 40 x 10 6 C57BL/6 cells on days l and 8 of the experiment. Three days after the second immunization 4 to 8 mice in each group were treated with IL2 by constant subcutaneous infusion, delivered by osmotic pumps implanted subcutaneously, 10 5 units/day for 14 days. Cohorts of mice were also simultaneously treated with etianate, 1.5 mg/day, also delivered by constant infusion. At the end of the experiment, mice were sacrificed, spleens removed and cellularity assessed. Spleen cells were then used as effector cells against EL4, a C57BL/6-derived leukemia line. Results shown in Table 5 represent the average total lytic units recovered per mouse. As the data in Table 5 demonstrate, administration of etianate resulted in a yield of greater lytic units against allospecific leukemia target cells when animals were co-treated with IL2; no augmentation of cyto¬ toxicity was seen, on the other hand, when the com¬ pounds were administered alone and animals did not receive IL2. These data confirm that etianate acts synergistically with IL2 in the cell-mediated immune response following in vivo allosensitization and suggest that IL2 plus etianate may also find a clinical application as a modality to boost effects of specific immunizations. As shown in Table 5, allospecific cytotoxicity, measured by the lysis of EL4 leukemia cells, was not decreased by etianate, was marginally increased by treatment with IL2 only (p = 0.054) and was substan¬ tially augmented by treatment of immune mice with IL2 plus etianate (p = 0.026). Furthermore, the serum chemistry profiles obtained from mice treated with IL2 and steroids showed that the only discriminant in all animals tested was hepatic function, whereas renal function, assessed by the determination of serum creatinine and BUN was normal in all treatment groups. IL2 induced hepatic dysfunction (total serum bilirubin 2.5 ± 1.3 mg/dl, p = 0.095). Mice treated with IL2 and hydrocortisone had a serum bilirubin level of 1.7 ± 0.6 (not significantly different from control or IL2-only) and those treated with IL2 plus etianate had normal bilirubin levels (1.3 ± 0.5 mg/dl, p = 0.48) . There were no differences in alkaline phosphatase levels. Glutamic oxalacetic transaminases were uniformly elevated, compared to controls, in mice treated with IL2 only and with steroids (p = 0.024). The glutamic-pyruvic trans- aminase was marginally elevated in the IL2 plus hydrocortisone group. Weight, as a general parameter of well-being, was increased by 30% over baseline in mice receiving IL2 only, and was not significantly different from that of mice treated with D5W (24%) , IL2 plus hydrocortisone (24.5%) or IL2 plus etianate (23.4%). Histological examinations of mice treated with IL2 were performed to determine the extent and nature of the effect of IL2 on various organs. Treatment with IL2 at a dose of 10 5 units daily, failed to demonstrate abnormalities. However, in mice treated at high-dose IL2 (8-10 x 10 5 units/day for 5 days) , which is uniformly lethal for BALB/c mice by day 6 of treatment, substantial pulmonary, hepatic and renal infiltration with mononuclear cells were found. However, the microscopic appearance of lungs, liver and kidneys of animals treated with IL2 plus etianate are remarkably different. The histological findings showed that the proximal cause of death in animals receiving high dose IL2 is likely secondary to severe pulmonary congestion. IL2 induced periportal hepatic and interstitial inflam¬ matory infiltrates with scattered eosinophilic degeneration of hepatocytes, with pyknotic nuclei, that were rarely surrounded by lymphoid cells. The splenic hyperplasia consequent to treatment with IL2 was often impressive and occurred also in mice treated with etianate; conversely, mice treated with hydrocortisone all developed hypoplastic spleens with decrease of the pulp and of follicles. Animals treated with IL2 plus etianate had no evidence of interstitial inflammatory reactions in the lung, liver and kidneys, in contrast with the typical mononuclear cell infiltrates induced by IL2 in the same organs. In vivo, hydrocortisone prevented the organ toxicity of IL2, but it also ablated its im unoaug- menting effects. In contrast, in mice treated with IL2 plus etianate, protection against IL2 organ toxicities occurred with preservation of its immunoaugmenting antitumoral effects. The prevention of hepatic and other organ toxicities induced by both steroids appeared to be strongly related to the inhibition of local inflammatory reactions, reminiscent of dysreac- tive immune conditions such as those caused by treatment with IL2. Example 9 Hepatoprotective Effects of Etianate by Constant Infusion During Treatment with IL2 in the Rat Hepatic dysfunction was also a prominent effect of treatment with IL2 in a rat model (Table 6) . Anti¬ inflammatory activity and normalization of hepatic chemistry profiles by etianate was confirmed in a rat model which more closely reproduced clinical schedules of IL2 immunotherapy. The results presented in Table 6 were obtained in the following manner. Serum bilirubin and hepatic enzymes (plus or minus standard deviation) levels obtained in male Sprague-Dawley rats after 5 days of treatment with IL2 and corticosteroids by constant infusion using osmotic infusion pumps (Alzet model 2ML1, Alza, Palo Alto, CA) . These pumps have an internal reservoir of 2 ml of which 1.7 ml are delivered over a 7 day period with a mean pumping rate of 10 μl/hr. Pumps were implanted subcutaneously 2 to 3 cm below the scapulae and were removed on day 8 of treatment under identical anesthesia. Cohorts of 3 rats in each experiment were implanted with pumps containing control diluent (5% dextrose in water) , recombinant human IL2 35 x 10 6 units/Kg/day, or IL2 in combination with hydrocortisone or etianate, 40 mg/kg/day. The results shown in Table 6 demonstrate that both hydrocortisone and etianate exerted protective effects against IL2-induced hepatic toxicity, which was mea¬ sured by the determination of serum bilirubin, alkaline phosphatase, alanine and aspartate aminotransferase. Hepatic toxicity was not completely abrogated, however, in animals co-treated with hydrocortisone as shown by the elevation of ALT. In the rat both hydrocortisone and etianate prevented the elevation of serum bilirubin typically seen after 5-7 days of IL2 infusion. Hydrocortisone, however, while preventing inflammatory reactions, caused hepatic dysfunction which was characterized by the uniform elevation of liver enzymes without hyperbilirubinemia and, histologically, by fatty degeneration. The serological and histological findings in rats treated with IL2 and etianate were superimposable with those of control-treated animals. Unlike what is commonly observed in cancer patients undergoing IL2 immunotherapy, there was no evidence of fluid retention in rats. There was a 14% weight loss, after 5 days of treatment, in rats treated with IL2 plus hydrocortisone (p = 0.008), contrasted with a 5% weight gain in those receiving IL2 plus etianate. The difference between cortisone and etianate was significant (p<0.001). Example 10 Effect of Different Schedules of Hydrocortisone and Etianate on the Elevation of Bilirubin, Induced by IL2 in the Rat To further examine the hepatic effects of hydro- cortisone and etianate in the rat the following experi¬ ment was performed. The results are presented in Table 7. In each experiment, matched cohorts of rats receiving IL2, 35 x 10 6 units/kg of IL2 daily for 7 days by constant subcutaneous infusion through osmotic pumps and were treated with hydrocortisone or etianate, 40/mg/kg/day. The steroids were administered by constant infusion through osmotic pumps, by daily bolus injection subcutaneously and by oral administration through drinking water (0.4 mg/ml) . Serum bilirubin levels shown were determined after 5 and 7 days of treatment. As shown in the second column, there was no difference between the amount of steroids assumed by the oral or the parenteral routes. The results presented in Table 7 indicate that the hepatoprotective effects of etianate and of hydro¬ cortisone were schedule dependent. Table 7 shows bilirubin levels of rats treated with constant infu¬ sion, bolus infusion or oral administration of hydro- cortisone and etianate during constant infusion of IL2. Not all routes and schedules of hydrocortisone were effective: bolus injection did not prevent hepatotoxicity while oral administration reduced serum bilirubin levels (p = 0.004). Etianate was effective only by constant infusion (p = 0.003). The daily dose of steroids assumed with drinking water (shown in Table 7 are the average daily doses calculated by the mea- surement of daily water intake) did not differ from the doses administered parenterally. Similar schedule- dependent effects were seen on the elevation of hepatic enzymes. In the rat both hydrocortisone and etianate prevented the elevation of serum bilirubin typically seen after 5-7 days of IL2 infusion. Hydrocortisone, however, while preventing inflammatory reactions, caused hepatic dysfunction which was characterized by the uniform elevation of liver enzymes without hyper- bilirubinemia and, histologically, by fatty degenera¬ tion. The serological and histological findings in rats treated with IL2 and etianate were superimposable with those of control-treated animals. In order to elucidate IL2-induced hepatic dysfunc- tion, livers were examined histologically at the end of treatment. As already seen in the mouse, extensive mononuclear cell infiltrates, with scattered eosino- philic hepatocyte degeneration were seen in animals treated with IL2 only. In those treated with IL2 plus hydrocortisone there was uniform, mild steatosis with glycogen accumulation but inflammatory infiltrates were absent. The hepatic architecture of rats treated with IL2 plus etianate was comparable to that of controls. Example 11 Hematologic Effect of Hydrocortisone and Etianate During IL2 Treatment by Constant Infusion in the Rat In the rat it was possible to determine other hematologic effects of both steroids. It was found that etianate (but not hydrocortisone) reversed thrombocytopenia, a common effect of IL2 in humans when the lymphokine is administered by constant infusion (Redondo et al. (1988)) and lastly, hydrocortisone (but not etianate) , caused severe lymphocytopenia and deple- tion of splenic lymphoid follicles. The effect of hydrocortisone and etianate on hematologic effects was induced by treatment of rats with IL2 by constant infusion as described in Example 4. The hematologic effects of hydrocortisone and etianate during constant infusion of IL2 in rats were obtained as described in Example 4 and are shown in Table 8. As shown in Table 8, IL2 induced thrombocytopenia and eosinophilia in the rat as previously reported for humans (Paciucci et al. (1990)). Etianate prevented IL2-induced thrombocytopenia, but not eosinophilia, when administered by constant infusion and not when given orally (p = 0.058 and p = 0.002 respectively). Hydrocortisone, on the other hand, did not prevent thrombocytopenia while causing severe lymphocytopenia by both the constant infusion and oral routes (p = 0.001 for each).- The two schedules of glucocorticoids failed to protect animals against IL2-induced thrombocytopenia and both caused lymphocytopenia (p = 0.001). Although etianate did not prevent eosinophilia, it did not cause lymphocytopenia and prevented thrombocytopenia.

Claims: Claims 1. A method of treating cancerous tumors in a human subject with IL2, comprising administering IL2 to the subject in dosage amounts sufficient to cause antitumor activity, and further comprising administering to the subject etianate or an agonist thereof in dosage amounts sufficient to reduce the toxic side effects otherwise associated with the administration of IL2, whereby said antitumor activity is obtained and said toxic side effects are reduced. 2. The method according to claim 1, comprising the step of administering etianate in dosage amounts sufficient to reduce the toxic side effects otherwise associated with the administration of IL2. 3. The method according to claim 2, including the step of orally administering etianate to the subject. 4. The method according to claim 2 , including the step of parenterally administering etianate. 5. The method according to claim 4, including the step of administering etianate intravenously. 6. The method according to claim 4, including the step of administering etianate subcutaneously. 7. The method according to claim 2, wherein the etianate is administered substantially con- tinuously throughout the period of administration of IL2. 8. The method according to claim 7, wherein the etianate is administered in multiple doses around the clock. 9. The method according to claim 7, wherein the etianate is administered by means of an infusion pump. 10. The method according to claim 2, wherein the etianate is administered in dosages comprising at least 1 mg during any given 24 hour period. 11. The method according to claim 10, wherein the etianate is administered in dosages comprising from about 1 mg to about 10 g during any given 24 hour period. 12. The method according to claim 11, wherein the etianate is administered in dosages comprising from about 10 mg to about 1 g during any given 24 hour period. 13. The method according to claim 12, wherein the etianate is administered in dosages comprising from about 100 mg to about 500 mg during any given 24 hour period. 14. The method according to claim 2, wherein at least one of liver toxicity, obstructive jaundice, renal dysfunction, capillary leak syndrome, oral mucositis, nausea, vomiting, protracted anorexia, diarrhea, fever, dermatitis, and mental dysfunction normally associated with the administration of IL2 is reduced while antitumor activity of IL2 is retained. 15. The method according to claim 14, wherein liver toxicity is reduced. 16. The method according to claim 14, wherein obstructive jaundice is reduced. 17. The method according to claim 14, wherein renal dysfunction is reduced. 18. The method according to claim 2, wherein the toxic side effects otherwise associated with the administration of IL2 are sufficiently reduced to enable the administration of IL2 to the subject in dosages at least as high as dosages that would otherwise be medically precluded by the toxic side effects. 19. An antitumor treatment composition comprising, in combination, IL2 in a dosage amount sufficient to cause antitumor activity in a human subject, and an amount of etianate or an agonist thereof sufficient to ameliorate at least one of the toxic side effects normally associated with the administration of IL2 to a patient. 20. The method according to claim 19, wherein the combination includes etianate. 21. A method of treating adverse health conditions in humans, said conditions being those which normally respond favorably to treatment with cortisone, said method comprising administering to the human a therapeutically effective amount of etianate. 22. A method of treating adverse health conditions in humans, said conditions being those which normally respond favorably to treatment with a biological response modifier having toxic side effects, said method comprising administering to the human the biological response modifier and an amount of etianate or an agonist thereof sufficient to ameliorate at least one of the toxic side effects normally associated with the administration of the biological response modifier. 23. The method according to claim 22 wherein the biological response modifier is selected from the group consisting of interleukins, transforming growth factor-r/S, thromboxane A2, α-2 granules and tumor necrosis factor. 24. The method according to claim 23 wherein the interleukins are selected from the group consisting of IL1, IL2, IL3, IL4, IL6 and IL8. 25. The method according to claim 22, comprising the step of administering etianate in dosage amounts sufficient to reduce the toxic side effects otherwise associated with the administration of IL2. 26. The method according to claim 24, including the step of orally administering etianate to the subject. 27. The method according to claim 24, including the step of parenterally administering etianate. 28. The method according to claim 26, including the step of administering etianate intravenously. 29. The method according to claim 26, including the step of administering etianate subcutaneously. 30. The method according to claim 24, wherein the etianate is administered substantially con- tinuously throughout the period of administration of IL2. 31. The method according to claim 29, wherein the etianate is administered in multiple doses around the clock. 32. The method according to claim 29, wherein the etianate is administered by means of an infusion pump. 33. The method according to claim 24, wherein the etianate is administered in dosages comprising at least 1 mg during any given 24 hour period. 34. The method according to claim 32, wherein the etianate is administered in dosages comprising from about 1 mg to about 10 g during any given 24 hour period. 35. The method according to claim 33, wherein the etianate is administered in dosages comprising from about 10 mg to about 1 g during any given 24 hour period. 36. The method according to claim 34, wherein the etianate is administered in dosages comprising from about 100 mg to about 500.mg during any given 24 hour period. 37. A composition for vaccination of mammals including man comprising an antigen, a pharmaceutically acceptable carrier therefor and an amount of etianate or agonists thereof and IL2 effective to boost the immune response of the mammal to the antigen. 38. A composition for treating autoimmune diseases comprising a pharmaceutically acceptable carrier and an amount of etianate or agonists thereof effective to ameliorate at least one symptom of the autoimmune disease. 39. A method of treating cancerous tumors in a human subject with IL2, comprising administering IL2 to the subject in dosage amounts sufficient to cause antitumor activity, and further comprising administering to the subject at least one of etianate or tetrahydrocortisone or agonists thereof in dosage amounts sufficient to reduce the toxic side effects otherwise associated with the administration of IL2, whereby said antitumor activity is obtained and said toxic side effects are reduced. 40. An antitumor treatment composition comprising, in combination, IL2 in a dosage amount sufficient to cause antitumor activity in a human subject, and an amount of at least one of etianate or tetrahydrocortisone or agonists thereof sufficient to ameliorate at least one of the toxic side effects normally associated with the administration of IL2 to a patient. 41. A method of treating adverse health conditions in humans, said conditions being those which normally respond favorably to treatment with hydro- cortisone, said method comprising administering to the human a therapeutically effective amount of at least one of tetrahydrocortisone or etianate. 42. A method of treating adverse health conditions in humans, said conditions being those which normally respond favorably to treatment with a biological response modifier having toxic side effects, said method comprising administering to the human the biological response modifier and an amount of at least one of etianate or tetrahydrocortisone or agonists thereof sufficient to ameliorate at least one of the toxic side effects normally associated with the administration of the biological response modifier. 43. A method of treating cancerous tumors in a human subject with IL2, comprising administering IL2 to the subject in dosage amounts sufficient to cause antitumor activity, and further comprising administering to the subject tetrahydrocortisone or agonists thereof in dosage amounts sufficient to reduce the toxic side effects otherwise associated with the administration of IL2, whereby said antitumor activity is obtained and said toxic side effects are reduced. 44. An antitumor treatment composition comprising, in combination, IL2 in a dosage amount sufficient to cause antitumor activity in a human subject, and an amount of tetrahydrocortisone or agonists thereof sufficient to ameliorate at least one of the toxic side effects normally associated with the administration of IL2 to a patient. 45. A method of treating adverse health conditions in humans, said conditions being those which normally respond favorably to treatment with hydrocortisone, said method comprising administering to the human a therapeutically effective amount of tetrahydrocortisone. 46. A method of treating adverse health conditions in humans, said conditions being those which normally respond favorably to treatment with a biological response modifier having toxic side effects, said method comprising administering to the human the biological response modifier and an amount of tetrahydrocortisone or agonists thereof sufficient to ameliorate at least one of the toxic side effects normally associated with the administration of the biological response modifier.

Inventors: HOLLAND JAMES F, PACIUCCI PAOLO ALBERTO, HOLLAND, JAMES, F., PACIUCCI, PAOLO, ALBERTO
Applicants: SINAI SCHOOL MEDICINE, MOUNT SINAI SCHOOL OF MEDICINE OF THE CITY UNIVERS
Application year: 1992
Main classification: C07K13
IPC codes: A61K31, A61K38, A61K39, A61K45, A61P35, A61P37, C07J3