WO1992016235A1 - Compositions and methods of treatment of a variety of disorders utilizing etianate and agonists thereof - Google Patents
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- WO1992016235A1 WO1992016235A1 PCT/US1992/002423 US9202423W WO9216235A1 WO 1992016235 A1 WO1992016235 A1 WO 1992016235A1 US 9202423 W US9202423 W US 9202423W WO 9216235 A1 WO9216235 A1 WO 9216235A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/191—Tumor necrosis factors [TNF], e.g. lymphotoxin [LT], i.e. TNF-beta
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/20—Interleukins [IL]
- A61K38/2013—IL-2
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
Definitions
- 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.
- 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
- 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.
- IL2 administered to animals and humans by constant infusion induces proliferation of natural killer (NK) cells and T lymphocytes.
- NK natural killer
- 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) .
- 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.
- Hydrocortisone (17 ⁇ 21-Dihydroxy-4-pregnane-3,ll, 20-trione) , noted for its broad-scale activities, is utilized widely in medicine for its anti-inflammatory activity.
- 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.
- 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.
- 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 (3 ⁇ ,17 ⁇ ,21-Trihydroxy- j8-pregnane-ll,20-dione) , the metabolic end product of hydrocortisone, is thought to be devoid of glucocorticoid activity.
- Tetrahydrocortisone in combination with heparin, has been found to possess anti-angiogenic 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).
- an unmeasured amount may be excreted in the bile.
- 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.
- 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
- 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.
- PHA phytohemagglutinin
- IL2 systemic and organ toxicity induced by IL2
- 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.
- platelets degranulate and release ⁇ -2 granules.
- Factors contained in ⁇ -2 granules include proteins with anti-cancer activity such as transforming growth factor ⁇ (TGFjS) and platelet factor 4 (PF4) and are found in the plasma of patients within a few hours of IL2 infusion.
- TGFjS transforming growth factor ⁇
- PF4 platelet factor 4
- the ability of etianate to ameliorate thrombocytopenia in IL2 treatment may be due to blockage of the effects of thromboxane A2, ⁇ -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.
- TNF tumor necrosis factor
- 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.
- 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, ⁇ -2 granules, TNF and natural or recombinant preparations of ⁇ , ⁇ and y interferons.
- interleukins such as IL1, IL3, IL4, IL6, PF4, TGFS, thromboxane A2, ⁇ -2 granules, TNF and natural or recombinant preparations of ⁇ , ⁇ and y interferons.
- 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.
- 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.
- hydrocortisone 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.
- hydrocortisone while preventing hyperbilirubinemia, caused extensive hepatic necrosis which was documented both histologically and serologically.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- etianate can be administered in dosages exceeding that of hydrocortisone.
- 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.
- 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.
- an effective level of etianate would be about 1% in a physiologically acceptable carrier.
- Administration of etianate compounds would cease once healing has occurred.
- etianate when 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.
- 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.
- Lymphocytes were gradient separated from hepar- inized peripheral blood of healthy volunteers and cultured 2xl0 6 /ml in RPMI 1640 medium (Gibco, Grand
- Emeryville CA Emeryville CA
- 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.
- 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.
- panel 2C cultures were pretreated with etianate for one hour and then with hydrocortisone before the addition of IL2.
- 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.
- mice 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.
- 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.)
- 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).
- 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.
- 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.
- 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.
- Thrombocytopenia a common effect of IL2 in humans when the lymphokine is administered by constant infu ⁇ sion, was also seen in rats (Table 1) .
- clear ⁇ ing of lymphocytes from the peripheral blood does not occur in rats during administration of IL2 by constant infusion.
- Absolute lymphocytopenia occurred in the group of animals receiving IL2 plus hydrocorti- sone in whom the average baseline lymphocyte count was 7510/ ⁇ l and decreased after 7 days of treatment to 2445/ ⁇ l (p ⁇ .0001 by Student's t test).
- lymphocyto- penia was attributed to the lymphocytotoxic effects of glucocorticoids.
- PHA phytohemagglutinin
- 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 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.
- % lysis (cpm test - cpm spontaneous release) x 100 (cpm maximum release - cpm spont. release)
- 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 ⁇ l) of cultured effector cells at 10 6 /ml were conjugated with each mAb (final concentration 2 ⁇ g/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) .
- 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.
- D5W control diluent
- IL2 IL2 only
- 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 ⁇ g/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.
- 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.
- 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)
- lytic units 20 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.
- mice 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.
- 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) .
- 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.
- 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).
- 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.
- mice Male BALB/c mice, 6 to 12 weeks old, were immunized with 40 x 10 6 C57BL/6 cells on days l and
- 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.
- 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%).
- mice treated with IL2 were performed to determine the extent and nature of the effect of IL2 on various organs. Treatment with IL2
- IL2 at a dose of 10 5 units daily failed to demonstrate abnormalities.
- mice treated at high-dose IL2 8-10 x 10 5 units/day for 5 days
- substantial pulmonary, hepatic and renal infiltration with mononuclear cells were found.
- the microscopic appearance of lungs, liver and kidneys of animals treated with IL2 plus etianate are remarkably different.
- 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 spectacular 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.
- mice 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.
- hydrocortisone prevented the organ toxicity of IL2, but it also ablated its im unoaug- menting effects.
- 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.
- 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 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.
- 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.
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JP4507897A JPH06505491A (en) | 1991-03-21 | 1992-03-20 | Methods of treating various diseases using etianate and its agonists and compositions used therein |
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US67302691A | 1991-03-21 | 1991-03-21 | |
US673,026 | 1991-03-21 | ||
US71799591A | 1991-06-20 | 1991-06-20 | |
US717,995 | 1991-06-20 |
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PCT/US1992/002423 WO1992016235A1 (en) | 1991-03-21 | 1992-03-20 | Compositions and methods of treatment of a variety of disorders utilizing etianate and agonists thereof |
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EP (1) | EP0581803A4 (en) |
JP (1) | JPH06505491A (en) |
CA (1) | CA2105378A1 (en) |
WO (1) | WO1992016235A1 (en) |
Cited By (1)
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EP3303636A4 (en) * | 2015-06-03 | 2018-11-21 | Aelan Cell Technologies, Inc. | Companion methods and kits for il-2-based therapies and mesenchymal stem cell-based therapies |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
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US4599331A (en) * | 1984-12-24 | 1986-07-08 | Syntex (U.S.A.) Inc. | Etianic acids as antiangiogenics |
US4876250A (en) * | 1988-10-31 | 1989-10-24 | Alcon Laboratories, Inc. | Methods for controlling ocular hypertension with angiostatic steroids |
US4945089A (en) * | 1987-12-29 | 1990-07-31 | Alcon Laboratories, Inc. | Use of tetrahydrocortexolone to prevent elevations in intraocular pressure caused by corticosteroids |
-
1992
- 1992-03-20 CA CA002105378A patent/CA2105378A1/en not_active Abandoned
- 1992-03-20 JP JP4507897A patent/JPH06505491A/en active Pending
- 1992-03-20 WO PCT/US1992/002423 patent/WO1992016235A1/en not_active Application Discontinuation
- 1992-03-20 EP EP19920908745 patent/EP0581803A4/en not_active Withdrawn
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4599331A (en) * | 1984-12-24 | 1986-07-08 | Syntex (U.S.A.) Inc. | Etianic acids as antiangiogenics |
US4945089A (en) * | 1987-12-29 | 1990-07-31 | Alcon Laboratories, Inc. | Use of tetrahydrocortexolone to prevent elevations in intraocular pressure caused by corticosteroids |
US4876250A (en) * | 1988-10-31 | 1989-10-24 | Alcon Laboratories, Inc. | Methods for controlling ocular hypertension with angiostatic steroids |
Non-Patent Citations (8)
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3303636A4 (en) * | 2015-06-03 | 2018-11-21 | Aelan Cell Technologies, Inc. | Companion methods and kits for il-2-based therapies and mesenchymal stem cell-based therapies |
EP4015652A1 (en) * | 2015-06-03 | 2022-06-22 | Aelan Cell Technologies, Inc. | Companion methods and kits for il-2-based therapies and mesenchymal stem cell-based therapies |
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Publication number | Publication date |
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CA2105378A1 (en) | 1992-09-22 |
JPH06505491A (en) | 1994-06-23 |
EP0581803A4 (en) | 1994-03-18 |
EP0581803A1 (en) | 1994-02-09 |
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