Note: Descriptions are shown in the official language in which they were submitted.
` W092/16235 PCT/US92~02423
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Description
COMPOSITIONS AND METHODS OF TREATMENT OF A V~R1ET~` ()F r)lSORDERS
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
Interleukin-2 (IL2), initially describéd as a
paracrine growth factor for T cells, induces
non-specific cell-mediated antitumor 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); Domzig 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",
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WO92/16235 PCT/US92/02423
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Nature, 302:305-310 (1983). The purified recombinant
~L2 has allowed exploration of the IL2 antitumor
activity in animals and humans. Kirkman et al., J.
Exp. Mad., 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.
onc., 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 ln vitro.
A positive relationship exists between IL2 dose-
intensity and frequency of response. Paciucci et al.,
"Immunotherapy with Interleukin-2 by Constant Inf~usion
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
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W092/1623~ PCT/US9~/02423
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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 (17~ 21-Dihydroxy-4-pregnane-3,11,
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,
hemolytic anemia, brain edema, fibrositis and shock.
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W092t~6235 PCT/US92/Q2423
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Hydrocortisone has also found ~reat 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 metastases of some cancers.
Tetrahydrocortisone (3~,17~,21-Trihydroxy-
~-pregnane-11,20-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
vlvo activity, tetrahydrocortisone has been found to
lack the immunosuppressive effect of hydrocortisone.
Langhoff et al., "The Immunosuppressive Potency n
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
WO92~16235 PCT/US92/02423
3~
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-~-
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 ViYo. Etianate therefore fulfills the
requirements for a candidate anti-inflammatory, non-
immunosuppressive ag2nt to be used in the clinic in
conjunction with biological response modifiers.
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WO92/16235 PCT/US92/0~23
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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 Drawinqs
Figure l 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, lO0 U/ml ~I.) in the presence of
0.5 mg/ml of hydrocortisone (F) or etianate (Et) for 24
(panel l) and for 72 hours (panel 3). Panel 2 shows
results of cultures exposed to steroids for l hr. and
then washed three times before addition of IL2.
Figure 2 is a set of graphs depicting total lytic
units20/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 l) and bilirubin
(panel Z) de~ermined one day after the tenth injection
of IL2, IL2 plus hydrocortisone (F), IL2 plus etianate
3S (Et~ or D5W.
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Detailed Descri~tion 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
in vitro and in vivo immunosuppre~sive 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 immunoaugmenting 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 prese.rved 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
W O 92/16235 ` PC~r/US9~`02423
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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 ~-2 granules.
Factors contained in ~-2 granules include proteins with
anti-cancer activity such as transforming growth factor
~ (TGF~) 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, -2
granules, TGF~ 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~, 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 ILl,
IL3, IL4, IL6, PF4, TGF~, thromboxane A2, ~-2 granules,
TNF and natural or recombinant preparations of ~, ~ and
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
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broad range of substances which are otherwise known to
be beneficial in the treatment of various dis~ases~
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 effectsO
The effects of equimolar concentrations of
hydrocortisone were compared to those of etianate on
the induction of lymphokine activated killer cells in
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
W O 92J1623~ P(~r/US92~02423
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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. In 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 eosinop~lil 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.
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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
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compounds are administered to the patient in an amount
sufficient to provide therapeutic levels of etianateO
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
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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, II: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 10 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.
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W092/16235 PCT/~S92~0~23
210~378 -14- ~
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 Lymphoc~tes Treated with IL2
Lymphocytes were gradient separated from hepar-
inized peripheral blood of healthy volunteers andcultured 2xl06/ml in RPMI 1640 medium (Gibco, Grand
Island Biologic Co.) supplemented with 20% fetal bovine
serum. Exogenous recombinant IL2 (Cetus Corporation,
Emeryville CA), W2S 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 s~Chromium release assay against
DND-lA, a melanoma cell line developed in our labora-
tories. Percent tumor lysis at different effector/
target cell ratios was converted into lytic Units 20%
(LU20) defined as the number of effector cells contained
in each culture that could lyse 20% of target tumor
cells.
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W092/16235 ~1 0~ 37 gCT!U~921~23
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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.
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Example 2
The Effect Of in vivo Administration
of Hydrocortisone And Etianate
Irl cs7BL/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 20tmouse 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, 105 units in 0. 2 ml of D5W daily for
f ive days and the control with o. 2 ml of D5W only.
After a 2 day interval, each group was randomized into
20 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
25 0 . 2 ml of D5W. Control mice received a second 5
day-treatment with D5W intraperitoneally ti.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
35 T test: p < 0.0001~ and EL4 (p = 0.008), whereas
etianate did not suppress IL2 dependent tumor cyto-
., . - . . .
.
WO92/16235 PCT/~92/0~23
~ -17-
21~378
toxicity. The difference between hydrocortisone and
etianate was significant (p = 0.006) for YAC and
p = 0.008 for EL4). Hydrocortisone significantly
s;uppressed 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 Toxicitv 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 withhydrocortisone (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
WO92/16235 PCTtUSg~02423
-18-
2~ 8
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 106 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 , 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).
. , .
,
. W~92/16235 PCT/US92/02~23
210~37~
L TABLE 1 l
TOXICITIES OF TREATMENT WITH I -
IL2 AND HYDROCORTISONE OR ETIANATE
IIN THE RAT TOXICITY GRADE
I lo 11 12 13 14
= _ = = - 0=>150,000/ul
Platelets CTR 3 2 _0 1 1 1= 100-150,000/ul
IL2 0 21 3 1 2= 75-100,000/ul
n=7 IL2/F 3 10 2 1 3=25-75,000/ul
__ _ 11
10IL2/Et 4 1 1 1 0 ~=~25,D00/
Bilirubin CTR 7 0 0 0 0 0= <1.5 mg/dl
IL2 1 1 0 5 0 1=1.2-1.8 mg/dl
n=7 IL2/F 7 0 0 0 0 2=1.9-3.6 mg/dl
IL2/Et 5 1 0 1 0 3= >3.6 mg/dl
ALT CTR 5 2 O O O 0= <40 U
IL2 0 0 5 2 0 1= 40-60 U ¦
n=7 IL2/F 0 0 3 4 0 2=60-120 U
_
IL2/Et 0 1 5 1 0 3= >240 U
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
W092/16~5 PCT/US~!0~23
2~378 ~ ,
:
a~ministration 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 1 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
.
.
. W092/~6235 -21- 21 ~ 5 3 7 ~ /US92/0~3
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 = O.S, 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/~1 and decreased after 7 days of treatment to
2445/~1 (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 ofglucocorticoids. An additional corroboration of
immunosuppression induced by hydrocortisone is the
finding of an 82% decrease of ln 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 in vitro proliferation
to PHA in the rats receiving ~W, 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
W092/16235 PCT/US92/02423
-22-
21~S3r~8
during treatment with I~2. Weight loss was worse in
animals receiving IL2 plus hydrocortisone, in whom the
a~erage weight after treatment was 81% of baseline
(E~ = 0.003). In contrast, on day 8 of treatment, the -
a~erage 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 obtainthe data presented in Tables 2-8 and the histological
data discussed in the Examples.
Lvmphocvte 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, M0). Both compounds were diluted with 5%
dextrose in water (D5W).
Short-Term Activation with Interleukin-2:
Recombinant human IL-2, generously provided by the
. ~
. W092/16235 PCT/~92/024~
~ -23- 21`~ ~3~$
Chiron Corporation, Emeryville, CA, was reconstituted
in D5W. Ten x lO6 lymphocytes in lO ml medium were
activated with IL2, 600 IU/ml in 25 cm2 upright culture
flasks (corning Glass Works, corning, NY). Cultures
were incubated for 24 hours at 37C in a humidified
atmosphere of 5% C02. 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, inGubated and washed three times prior to the
addition of IL2.
Mixed Lym~hocyte Cultures: Five x 106 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 allosensitiæation and
steroid treatment, upon termination of cultures, O.l ml
aliquots of each were labeled with 0.2 ~Ci 3H-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 l900-CA beta
counter.
CYtotoxicit~: After harvesting, cells were
washed, suspended at 2 x lo6 cells/ml and O.l ml
'
,
,~ ' ' ' . .
WO92/16235 PCT/US92/0~23
210~3'~8 -24- ~ '
aliquots dispensed in round-bottom 96-well microtiter
plates together with 0.1 ml containing 5 x 103 target
cells that had been labeled with 100 ~Ci of 5ICr (New
h'ngland Nuclear, MA). In each experiment, 3
~ffector/target cell ratios were studied (40:1, 10:1
and 2.5:1) in 4 replicate wells. Microcytotoxicity
trays were incubated at 37C in a humidified atmosphere
of 5~ C02 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 lN 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% (LU20), defined
as the number of effector cells contained in each
culture sample that could lyse 20% of 5 x 103 target
cells.
Immunop~henotypina: Monoclonal antibodies (mAb)
Leu4 (CD3), Leul2 (CDl9) and anti-IL2 receptor (CD25),
conjugated with either fluorescein or phycoerythrin
were obtained from Becton-Dickinson. Aliquots (150 ~l)
of cultured effector cells at l06/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 l~ para-
formaldehyde. Cell subpopulations were defined by the
backgating procedure using the simultest Leuko-Gate
staining technique and Simultest software according to
the manufac~urer's instructions (Becton-Dickinson).
Cells were analyzed for differential staining patterns.
.
.. . . -- -- .
. .
- . . . . .
.. .. . . .
. W092/~6235 - PCT~US~0~23
~ -25- ~10~378
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 kilier
cells with IL2 and steroids. Mice were inoculated
subcutaneously with IL2, 6 x 105 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, glutamic-pyruvic and glutamic-
oxalacetic transamina~es).
In vivo Alloaeneic Sensitization: BALB/c mice
were immunized twice in an 8 day period with 40 x Io6
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 Taraet 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
' :~. . - , ' : . `
: ~' ' ; : '' ' ~
W092/~235 PCT/US92/024~3
-26-
5 3 7 8
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, ~S), 12 mg/kg
intramuscularly and ketamine (Ketalar, Parke-Davis,
Morris Plains, NH), 30 mg/kg intraperitoneally 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.
In each experiment cohorts of three rats were
treated with constant infusion of control diluent
(D5W), IL2, 29 x 106 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 in ~itro generation of anti-tumor
:. .. . -
. WO92/1623~ PCT/US~2/02423
~ -27- ~ 0~378
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. Effectorcells (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, TL2-induced activity was tested against a
2S 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).
:: . . . -
, ' .:
: ., .'. '' :
WO92/1~23~ PCT/US92/0~3
-28-
2iQ~7`8
j TABLB 2
v ~, l I
EFFECT OF HYDROCORTISONE (F) AND ETIANIC ACID (Et)
ON IL2-INDUCED CYTOTOXICITY AND IL2-RECEPTOR
EXPRESSION IN HUMAN LYMPHOCYTES
IL2, 600 IU/ml LU~o x CD3- ~ CD3+
10 , p** CD3+ CD25+ CD25+
Average*
a. ControlS18 A. 71.7 17.8 11.5
b. F 0.05 95 .010 B. 66.5 20.3 14.9
mg/ml x 24
c. Et 0.05601 .025 C. 73.0 lS.8 11.5
mg/ml x 2
. _
d. F x lh188 .Q14 D. 69 5 17 9 11 6
11
e. Et .25692 .023 E. 70.8 16.6 11 1
mg/ml x lh
f. Et .25309 .037 F. 71.1 17.0 11.4 ¦
mg/ml x lh
then F .05
mg/ml
d. vs. f. B. vs. A. .023 .037 .014
p=.014**
e. vs f. B. vs. C. .014 .058 .016
*: average of 6 experiments
**: Wilcoxon Signed Rank Test
LU20 x 106: lytic units per million of effector cells
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 unitsrecovered 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
.
.
.
.
,
,:
`. WO92/lfi235 PCT/~ 2~0~423
~, -29-2,~3~ ~ ~
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 = O.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 in vitro system, consequent to
lymphocytotoxicity. ~.he 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.
~xamle 6
Effect of Hydrocortisone and Etianate
on the Generation of Allospecific and
MHC-unrestricted Cytotoxicity
Followina in vitro Allosensitization
Lymphocyte allosensiti~ation generates effector
cells that are cytotoxic not snly for allospecific
target cells but also for allogeneic and syngeneic
. ~ . ~ - . . .
: ~ . . . : ,.
,
W092/~6235 PCT/US92/~23
-30- ~ .
210S378
tumor 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 ln 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 in 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 alO
(1983). The following experiment was designed to test
the effects of hydrocortisone and etianate on this
phenomenon.
The results shown in Table 3 were o~tained as
follows. Peripheral blood lymphocytes from healthy
volunteers were allosensitized for 7 days at 37C in a
humidified atmosphere of 5% CO2 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 5~Cr labeled allospecific normal target cells
and ~n unrelated melanoma cell line.
. - - .. ~
- ,:
. ' . ':
.
W092/16235 PCTtUS~ ~23
~ -31-
2i05378
Cytotoxicity assays were performed at diffèrent
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 lN HCl. Cytotoxicity
results were then expressed as lytic units20, in which a
lytic unit is the number of effector cells, for each
effector cell population, capable of lysing 20% of
target cells.
. .
: : : ' , . . ' -
.: .
.. :. : . . .. - .. ..
.. . .
--
W092/16235 PCT/US92/02423
!, . ~ ', ~ i _ 32
2i05378
¦ TABL~ 3
EFFECT OF HYDROCORTISONE (F) AND ETIANATE (Et) ON THE
GENERATION
OF ALLOSPECIFIC AND MHC-UNRESTRICTED CYTOTOXICITY
5FOLLOWING IN VITRO ALLOSENSITIZATION
LU20 X 106
TARGET CELLS
Culture Aver. B BMELANOMA CELLS
Treatment c.p.m IL2+ IL2
¦ p* ! l l I P*
AAx 891 0 0 - ! 0-5 14.7¦ 014
Control71328 78187.014 1 130 64I 014
ABx F 0.05 134512 3<.oool! 3 1 <.0001
ABx F 0.2619 1 5<.0001! 0 0 <.0001
mg/ml
ABx Et86942 112299NS ! 109 241 .014
0.05 mg/ml
ABx Et 0.2 5078971 119 NS ! 28 121 .014
mg/ml
Control NS .058 NS .037
mg/ml
_
Cgn/tltol 2 _ 03 NS .037 .014 =
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,
WO92/16235 PCT/US92/0~423
~33~ 21 ~ 53 ~8
however, the lytic ability of effector cells against
unrelated tumor cells. Whereas short-term incubation
~ith 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 in 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 mgtml (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
- , '
WO92/16235 PCT/US~/02423
-34-
210`~78 ``
effector cells to IL2 augmented cytotoxicity against
both allospecific and tumor target cells.
_xample 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 n 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 105 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 5~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.
` W092/1623~ PCT/US92~Q~4~3
~ r 7 8
,
TABLE 4
¦ IN VIVO EFFECTS OF STEROIDS ON THE GENERATION OF TUMORCYTOTOXICITY AGAINST SYNGENEIC AND ALLOGENEIC TUMORS
FOLLOWING IL2 AND ON THE GENERATION OF ALLOSPECIFIC
¦ CYTOTOXIC RESPONSES FOLLOWING ALLOIMMUNIZATION
T AVERAGE LU20/MOUSE
TARGE~ ' CELLS
L Exp. #1 Exp. #2
P20 EL4 YAC EL4
I ~
¦C57BL/6N*=10 N=10 N=8 N=4 ll
~I
¦D5W Control0 0 206 0 ¦¦
IL2 3712 3813 I10634984 ¦
IL2/F 96 13 2759 821
IL2/Et6467868 126253237
Significance**
IL2 vs. IL2LF 053 034<.0001 008
IL2 vs. IL2/Et131 211 634 197
IL2/F vs..140.003 .015.012
*: number of mice in each experiment
**: Student's t Test
D5W: 5% dextrose in water (diluent for IL2, F and Et)
The data presented in Table 4 indicate that in
C57BL/6 mice treated with IL2, 105 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
- ' : . . .
.
. . . . .
W092/16235 PCT/U~g2~0~23
210~8 -36- ~
.. . .
with mock-IL2 (126 and 145 x lo6, respectively).
Significantly higher recovery was always obtained in
those mice treated with IL2 plus etianate (161 x lo6,
p = 0.05) while the opposite was true for those treated
with IL2 plus hydrocortisone (105 x lo6, 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 ln vitro and ln 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).
Effect of Etianate on Allospecific Lysis
of EL4 by Spleen Cells of BALB/c Mice
Immunized Aqainst C57BL/6
To determine the ln 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 106 C57BL/6 cells on days 1 and
8 of the experiment. Three days after the second
W092~6235 PCT~lS~J~23
~ -37-
`` 2105378
immunization 4 to 8 mice in each group were treated
with IL2 by constant subcutaneous infusion, delivered
by osmotic pumps implanted subcutaneously, 105 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.
TABLB 5
EFFEC~ OF ETIANATE ON ALLO8PECIFIC LY8I8
OF EL4 ~Y 8PLEEN CELL8 OF BALB/C MICE
IMM~NIZED AGAIN8T C57 BL/C
~ i . _
Treatment Total LU20/ ¦ Student's
mouse EL4 ¦t Test
CTR 1743Control vs. Et 0.778 ¦¦
Et ~ 1914 Control vs. IL2 0.470 ¦¦
IL2 2134 IL2 vs. Et 0-054 l
__
IL2/Et 4654 Control vs. IL2 0.026 ¦
.
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 co,m-
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
~- . - '
. . - ~ . ~ . . .
,
, , : , ~ . ,
'" " ' '''' "' ' ''` .''' '
-
WO92/16235 PCT/US9~/0~23
-38-
210~37~
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
(24.55~) 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 105 units daily, failed to demonstrate
abnormalities. However, in mice treated at high-dose
IL2 (8-10 x 105 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
WO92/16235 PCT/US9~02423
~ - 2I0~378
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 immunoaug-
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.
- - - : . . .
.
, :. ~. ' . : . .
', ' ' ' ,,:
WOg2/16235 PCT/US~ 2423
2~1~S3~8.
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 ~l/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 Io6 units/Kg~day, or IL2 in
combination with hydrocortisone or etianate, 40
mg/kg/day.
WO92/16235 PCT/U~fO2423
-41- 2~ o~378
.. .
i
HEPATOPROTECTIVE EFFECTS OF ETIANATE BY CONSTANT
INFUSION DURING TREATMENT WITH IL2 IN THE RAT
. , ...... ... . .
N Treat- ¦Bilirubin ¦Alk Phos ALT AST
ment Img/dl
.
~ CTR 0.01 (0.03)247 (89)47 (16) 185
¦24 IL2 4.1 (3.1) 547 516 1]75 ¦
~ (193)(249) (552)
¦ 8 IL2-F 0.02 (0.06) 324 598 606 ¦
I (204)(748) (696)
¦12 IL2-Et 0.29 (0.57) 282 180 452 ¦
l (110)(174) (373) 1
_ . .
Unpaired Student's T test
. . ;
10 l IL2 vs. IL2-Et IL2 vs. IL2-F ¦¦
Bilirubin 0.001 <.0001
I 11
¦Alk. Phos. 0.030 .0003
I . 11
¦ALT 0.001 NS ¦¦
AST 0.001 0.010
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
,
,
. :
W O 92/16235 PC~r/US9~/02423
-42-
210~378 ;.
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 I~2 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 ~urther 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 106 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.
~ W092/16235 . PCT~US92/0~23
~ ~43~ 2105378
` - - I
TABLE 7 ¦
~ 1 I
EFFECT OF DIFFERENT SCHEDULES OF HYDROCORTISONE
AND ETIANATE ON THE ELEVATION OF BILIRUBIN,
INDUCED BY IL2 IN THE RAT ~-
5 _ N Avg. Bili- P Bili- _
mg/kg rubin4 rubin
Day 5 _ Day 7
F, SQ3 2 40 2.15 ~NS6 5.4 NS
IL2 control _ 5.7 9.5
F, p.o.2 9 37 0.04 0.004 0.04 0.003
IL2 control 2.37 3.54
10 IL2-F, C.I.78 40 0.01 0.0006 0.09 0.055
IL2 control 3.74 3.03
IL2-Et, 4 40 6.0 NS 2.53 NS
bolus 4.38 7.48
IL2 control
15 IL2-Et, P.O.6 32 2.8 NS 3.21 NS
IL2 control 4.63 3.29
IL2-Et 14 40 0.29 0.003 0.09 0.022
IL2 control 4.1 3.03
~ bolus = 40 mg/kg by bolus subcutaneous injection,
once daily
2 pO = O . 4 mg/ml of drinking water
3 SQ = 40 ~g/kg/day by bolus subcutaneous injection
once daily.
4 in mg/dl
5 statistical significance, by Wilcoxon Rank Test
6 NS = not significant
7 CI = 40 mg/kg/day by constant subcutaneous infusion
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
eff~ctive: bolus injection did not prevent
hepatotoxicity while oral administration reduced serum
.
.. .
W092/1~235 PCT/US92/02423
-44-
210S378
bilirubin levels (p = 0.004). Etianate was effective
only by constant infusion (p = 0.003). The daily dose
of steroids assumed with drin~ing water (shown in Table
7 are the average daily doses calculated by the mea-
surement of daily water intake) did not differ from thedoses 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 oftreatment. 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.
Exam~le 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
WO92/1623~ PCT/US~2/024~3
~ -45-
21~a378 `
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 ofhydrocortisone 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.
WO92~1623s PCT/US92/0~423
2 1 a~37 ~ ~ -46~
TABLB 8
DURING IL2 BY CONSTANT INFUSION IN THE RAT
- Day 5 Day 7 Day 7
N Platelets/ Lymphocytes/ Eosinophils/ ¦
1- -- ~1 ~1 ,ILl
¦ CONTROL 7 819,0003926 67
IIL2 only24 105,0005030 165
l I
¦IL2/F-P 7 144,000829 9
¦IL2/F-W 11 129,0001454 78
¦IL2/Et-P 13 394,0005408 152
¦ IL2/Et-W 6 113,0005262 227
; _ . ;l
¦Student's T
¦Control vs. (-000l 0.279 0.037 ¦¦
l IL2/vFsw 0.058 0.001 0.0S8
IL2/F-P 0.004 0.001 0.004
l IL2 vs. 0.002 0.423 0.143
l IL2/Et-P
.
Control vs. 0.073 0.755 0.34
IL2/Et-P
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).
. W092/1623~ PCT/US92J02423
~ ~47~ 210S378
The two schedules of glucocorticoids failed to
protect animals against IL2-induced thrombocytopenia
and both caused lymphocytopenia (p = O.OOl). Although
etianate did not prevent eosinophilia, it did not cause
lymphocytopenia and prevented thrombocytopenia.
" , ~
