Note: Descriptions are shown in the official language in which they were submitted.
W0 93~12789 PCI/US92/112~s
~ l 2 6 6 7 ~
~SE OF 5PIPERONE OR SPIPERONE DERIVAT~VES
AS IMMUNOSUPPRESSANT AGENTS
Backgrou~d of the Invention
This invention is in the field of the
suppression of immune responses, and in particular
provides a method for the treatment of immune
- disorders that preferably includes administering an
effective amount of spiperone or a spiperone
derivative, or a pharmaceutically acceptable salt,
including a quaternary salt, systemically or
topically.
The immune system specifically recognizes and
selectively eli~inates foreign invaders, or other
antigenic agents, by a process known as the immune
response. The immune response has three major
characteristics: it responds adaptively to foreign
invaders, it exhibits strong specificity, and it
displays a long-term memory of earlier contacts with
specific foreign pathogens or antigens. The immune
response involves the production of antibody and/or
the destruction of antigenic cells by lymphocytes,
which are highly specific ~or the antigen or hapten.
Cutaneous contact hypersensitivity responses are
complex expressions of a cellular immu~e response
characterized by antigen-dependent changes in
lymphocyte traffic, the recruitment of circulating
'~ukocytes to the site of antigen challenge (leukocyte
infiltration) and alterations in vascular permeability
and blood flow resulting in tissue swelling (edema).
In humans and companion animals, cutaneous conta~t
Ihypersensitlvi~ty responses can occur on exposure to
certain plant resins, such as those of poison ivy, and
other commonly encountered agents in the environment.
In individuals sensitized to such commonly encountered
agents, a severe contact reaction can result upon
exposure, with significant associated morbidity.
Severe or repeated contact hypersensitivity reactions
can~be followed by significant chronic changes, such
O g3/1~78~ 8 PCT/US92/11205
as scarring of affected tissues, itchines~, swelling,
scaling and oozing of tissue fluid through the skin
surface. This pathology may predispose the patient to
bacterial superinfection. In the eye, chronic immune
responses can lead to diminished vision or actual
blindness. In the lung, chronic immune responses,
such as chronic allergic asthma, can result in serious
chronic lung disease.
Cutaneous contact hypersensitivity and ~sthma
are just two examples of topical immune responses that
can be associated with significant morbidity. Others
include atopic dermatitis, ecæema, psoriasis,
Sjogren~s Syndrome, including keratoconjunctivitis
~-~ sicca secondary to Sjogren's Syndrome, alopecia
areata, allergic responses due to arthropod bite
reactions, Crohn's disease, aphthous ulcer, iritis,
conjunctivitis, keratoconjunctivitis, ulcerative
colitis, lichen planus, asthma, allergic asthma,
cutaneous lupus erythematosus, dry eye associated with
Sjogren's Syndrome, scleroderma, vaginitis, proctitis,
and drug eruptions. These conditions may result in
any one or more of the following symptoms or signs:
itching, swelling, redness, blisters, crusting,
ulceration, pain, scaling, cracking, hair loss,
scarring, or oozing of fluid involving the skin, eye,
or mucosal membranes.
In atopic dermatitis, and eczema in general,
immunologically mediated leukocyte infiltration
(particularly infiltration of mononuclear cells,
lymphocytes, neutrophi~s, and eosinophils) into the
skin importantly contributes to the pathogenesis of
these diseases. Chronic eczema aiso is associated
with significant hyperproliferation of the epidermis.
Similarly, psoriasis, a common cutaneous disease
associated with a hyperproliferating epidermis, also
has a leukocyte infiltration component.
Immunologically mediated leukocyte infiltration also
W093/12789 2 ~ 2 6 6 rl ~ PCT/US92~112V5
occurs at sites other than the skin, such as in the
airways in asthma and in the tear producing gland of
the eye in keratoconjunctivitis sicca.
In addition to disorders that clearly represent
S pathological consequenGes o~ immune responses, immune
responses are thought to contribute to many other
pathological conditions, including Crohn's disease and
ulcerative colitis of the gastrointesti..~l tract
(inflammatory bowel disease), psoriasis, alopecia
lo areata and others. While the cause o~ most of these
disorders is unclear, it is thought that exogenous
agents yet to be defined or components of the host's -:
own tissues (in the case of autoimmune disorders~ may
~-~ provoke an i~mune response that is responsible for the
infiltration of lymphocytes, monocytes, and
granulocytes observed in these conditions. It is also
believed that the infiltrating cells significantly
contri~ute to the tissue pathology associated with
these disorders, through the production of cytokines
as well as other mechanisms.
The need to control the wide variety of
pathological responses with immunological components
which result in cutaneous, ocular, or mucosal `~
hypersensitivity reactions, hyperproliferation, and
scarring has led to a search for effective therapeutic
agents that are both safe and effective.
Because of the importance of leukocytes and
their products in the development of pathology
assoc~ated with immune responses, many approaches to
~0 treating these conditions are focused on inhibiting
! the immune responses and leukocyte infiltration
contributing to these disorders. Several substances
are known to be able to inhibit the immune responses
contributing to cutaneous leukocyte responses or
hyperproliferative responses. Corticosteroids, when
administered systemically, are effective in this
regard but are associated with significant and
W093/1~789 2 ~ 2 '} ~ 7 8 PCT/ us 92/1 1205
potentially dangerous side effects. Topically applied
corticosteroids have some efficacy in treating these
conditions, but are only partially effective in many
instances and have their own significant side effects,
including atrophy of tissue, formation of
telangiectasia, blanching, and a myriad of systemic
effects if significantly absorbed. Other agents with
partial utility for treating some of the above
conditions include psoralen plus ultraviolet A (P WA),
cyclosporin A, or azathioprine, but the risk-to-
benefit ratios for these ag~nts is unfavorable for
most of the conditions described above.
As a resuit, there is a significant and very
~-~ long-standing need to identify new agents with
lS favorable benefit to risk ratios that can be applied
topically to prevent or suppress (i.e. "treat") immune
responses contributing to cutaneous, ocular, or
mucosal hypersensitivity reactions,
hyperproliferation, or scarring. Optimally, such
agents should be effective when applied locally, and
systemic absorption should not result in blood levels
high enough to cause significant systemic toxicity or
other adverse side effects. Not only does local
administration place the agent in closest contact with
the site needing treatment, but it also diminishes the
possibility that such treatment will suppress
beneficial immune responses which may occur at other,
more distant, sites.
Examples of pathogenic or undesired systemic
immune responses include host rejection of foreign
organ or tissue transplants; graft-vs-host disease in
which donor immunological cells present in the graft
attack host tissues in the recipient of the graft;
diseases with proven or possible autoimmune
components, such as rheumatoid arthritis, juvenile
rheumatoid arthritis, psoriatic arthritis, psoriasis,
leprosy reversal reactions, erythema nodosum leprosum,
W093/12789 PCT/U~g2/11205
212-~78
autoimmune uveltis, multiple sclerosis, allergic
encephalomyeliti5, systemic lupus erythematosis, acute
necroti~ing hemorrhagic encephalopathy, idiopathic
bilateral progressive sensorineural hearing loss,
aplastic anemia, pure red cell anemia, idiopathic
thrombocytopenia, polychondritis, scleroderma,
Wegener's granulomatosis, chronic active hepatitis,
myasthenia gravis, Stevens-Johnson syndrome,
idiopathic sprue, lichen planus, Crohn's disease,
Graves ophthalmopathy, sarcoidosis, primary biliary
cirrhosis, primary juvenile diabetes, dry eye
associated with Sjogren's syndrome/ uveitis posterior,
and interstitial lung fibros_-; allergic asthma; and
~-~ inappropriate allergic responses to en~ironmental
s~;.muli such as poison ivy, pollen, insect stings and
ain foods, including ~pic dermatitis and contact
.at
~ ~ous ther~?eutics that have been utilized a~
s~temic immunosuppressants include steroid hormones,
ZO anti-metabolites such as methotrexate and
azathioprine, cyclosporine, alky ~ting agents such as
cyclophosphamide and busulfan, and certain
antibiotics. However, there still remains a strong
need to provide new systemic immunosuppressive agents
that minimize or prevent these pathogenic immune
responses.
In contrast to the immune response, an
irflammatory response is a pathologic condition that
cc occur in response to immunologically non-specific
in,ury, either from physical ~such as trauma),
'chemical, or biologic agents. An inflammatory
response is characterized by increased blood flow ar.
redness in the inflamed area, increased capillary
permeability and edema, and recruitment of
immunologically non-specific white blood cells,
especially neutrophils, that remove injurious material
and promote repair. Unlike immune responses,
W093/12789 21~6!j7~ PCI`/US92/1~2(~5
inflammatory responses do not respond adaptively to
the inciting stimulus, do not show specificity and do
not exhibit long term memory.
Cellular products of lymphocytes may contribute
S to or induce an inflammatory response. However,
because of the dif~erences in mechanisms, a compound
can function as an antiinflammatory agent without
having immunosuppressive properties. Phenylbutazone,
indomethacin, aspirin, ibruprof~n, and acetaminophen
lo are examples of antiinflammatory compounds which have
no significant immunosuppressive activity, as
demonstrated by their lack of a significant effect on
immunological mediated responses, such as contact
~ hypersensitivity.
Spiperone (8-[3-{p-fluorobenzoyl}propyl]-l;
propyl]-1-phenyl-1,3,~-triazaspiro-~4.5~decan-4-one)
is a neuroleptic agent with central nervous system
(CNS) dopamine and serotonin (5-HT) receptor
antagonist properties. Some analogues of spiperone
are useful as experimental reagents in dopamine and
serotonin receptor studies. For example, the high
affinity of an immobilized spiperone derivative, 3-(2-
aminoethyl)-8-[3-(4-fluorobenzoyl)propyl]-4-oxo-1-
phenyl-1,3,8-tria~aspiro[4.5~decan-4-one
trihydrochloride, for dopamine receptors has made it
possible to isolate these receptors in pure form.
Radiopharmaceuticals based on spiperone and its
analogues have been shown to be useful in assessing
dopamine receptor function based on positron emission
tomography (PET) in animals and man. Spiperone has
also been shown to bind to human and mouse
lymphocytes, although the mechanîsm responsible for
such bindin~ is uncertain~
U.S. Patent No. 3,996,363 to Wade and U.S.
Patent No. 4,839,342 to Ooms, et al. disclose methods
to promote wound healing that include the application
of spiperone or a derivative thereof. Wound healing
WO93/12789 2 12 6 5 J ~ PCT/US92/11205
is a reparative process by which several types of
resident cells, such as epithelial cells, fibroblasts
and vascular endothelial cells, and certain
circulating cells~ including neutrophils, lymphocytes
and macrophages, act in concert to restore to a more
healthy condition tissues that have sustained various
forms of mechanical or other injury. Although
lymphocytes and macrophages participate in both wound
healing and in immune responses, the specific roles of
these cells in the two types of processes may be
distinct. In fact, treatment of wounds with
immunosuppressive agents, such as corticosteroids and
cyclosporin A~ has been known to cause impairment of
~ the healing pr~ess (Arch. Surg. May 1990, 125(5),
63F'-40; Ann. Ophthamol. April 1985; 17(4), 238, and J.
R ,. June 1983; 34(6), 572-5~. Therefore, a
nod to treat patient for wound healing does not
render obvious a ?ethod to immunosuppress that
patient.
There remains a need for compounds and methods
for the treatment of patients in need of topical or
systemic immmunosupression.
It is therefore an object of the present
invention to provide a method and compositions for the
topical or systemic suppression pathogenic immune
responses.
8ummary of the Invention
A method for the topical or systemic
limmunosuppression of a human or other mammal in need
of immunosuppression is disclosed wherein the mammal
is treated with an effective amount of spiperone or a
spiperone derivative, or its pharmaceutically
acceptable salt, optionally in a pharmaceutically-
acceptable diluent or carrier for systemic or topical
appl~cation.
WO93/12789 PCT/US92~1120~
:~2~5'7$ - :
The parent spiperone has a strong neuroleptic
effect when administered systemically, but not when
administered tcpically. It is used in the examples as
a model of an active immunosuppressant. In a
preferred embodiment for systemic treatment, a
spiperone derivative, or its pharmaceutically
acceptable salt, or a salt of the parent spiperone
that does not have a significant neuroleptic effect is
administered.
Spiperone or its derivative or pharmaceutically
acceptable salt is a preferred immunosuppressant if it
suppresses the leukocyte infiltrate and/or the ear
swelling associated with an experimental contact
~ hypersensitivity response by at least 40% at 24 hours
after specific antigen challenge.
In the preferred method of systemic
administration, the active compound is administered,
for example, by injection, in a pharmaceutical carrier
such as saline, in an amount effective to
immunosuppress the patient. In a second embodiment,
the derivatives are administered topically in a
suitable carrier to effectively immunosuppress the
patient at the site of application, without producing
a significant neuroleptic effect. Other
pharmaceutical compositions include a spiperone
derivative combined with a cycloamylose, such as
cyclodextrin, which can be used to modify the
pharmokinetics of the compound.
Spiperone and its active derivatives are useful
as topical agents in treating contact dermatitis,
atopic dermatitis, eczematous dermatitis, psoriasis,
Sjogren's Syndrome, including keratoconjunctivitis
sicca secondary to Sjogren's Syndrome, alopecia
areata, allergic responses due to arthropod bite
reactions, Crohn's disease (inflammatory bowel
disease), aphthous ulcer, iritis, conjunctivitis,
keratoconjunctivitis, ulcerative colitis, asthma,
W093/1~789 PCT/US92/1120~
2 ~ 7 ~
g
allergic asthma, cutaneous lupus erythematosus,
scleroderma, vaginitis, proctitis, and drug eruptions.
The novel method may also be useful in reducing the
infiltration of skin by malignant leukocytes in
diseases such as mycosis fungoides. These compounds
can also be used to treat an aqueous-deficient dry eye
state (such as immune mediated keratoconjunctivitis~
in a patient suffering therefrom, by administering the
compound topically to the eye.
Brief Description of the Figure~
Fiqure 1 - Effect of systemic spiperone (30 or
150 mg/kg, su~cutaneously) on the tiss~e swelling
associated Wit~l oxazolone-induced cutaneous contact
hypersensitivity reactions. Spiperone or vehicle
alone (0) was administered to C57BL/6J mice 1 hour
after challe~ for contact hypersensitivity. The
change in ea~ nickness (post-challenge value minus
baseline pre-challenge value) was measured 24 hours
after oxazolone challenge. The data are presented as
the mean + SEM (standard error of the mean). The
reduction in ear swelling observed with either 30 or
150 mg/kg spiperone was significant when compared to
the reactions observed in the control animals
( * *=p<O . O 1 ) .
Fiaure 2 - Effect of systemic treatment with 30
or 150 mg/kg spiperone/ subcutaneously, on leukocyte
in~iltration associated with 24-hour contact
hypersensitivity reactions. These data ~mean + SEM)
iare derived from the same mice whose ear t~ickness
values are shown in Figure 1. The reduction in
leukocyte infiltration observed in animals treated
with 30 or 150 mg/kg spiperone was significant when
compared to the reactions observed in animals treated
with vehicle alone (* or **=p<0.05 or 0.01,
respectively).
W O 93~12789 PC~r/US92/11205
~2'~7X
~iaure 3 - comparative effects of systemic ~
vehicle (1), haloperidol (2), trazadone (3), mianserin ;
(4) or spiperone (5) (all agents at 40 mg/kg,
subcutaneously) on the tissue swelling associated with
oxazolone-induced cutaneous contact hypersensitivity
reactions. Spiperone, the other agents, or vehicle
alone were administered to BALB/c mice 1 hour after
challenge for contact hypersensitivity. The change in
ear thickness (post-challenge value minus baseline
pre-challenge value) was measured 24 hours after
oxazolone challenge. The data are presented as the
mean + SEM. The reduction in ear swelling observed
with spiperone was significant when compared to the
~ reactions observed in the control, vehicle treated
animals (**=p<0.01), whereas haloperidol, trazadone
and mianserin did not significantly suppress the
tissue swelling associated with contact
hypersensitivity.
Fiaure 4 - Comparative effect of systemic
treatment with vehicle (1) or haloperidol (2),
trazadone (3), mianserin (4) or spiperone (5) (all
agents at 40 mg/kg), administered subcutaneously, on
leukocyte infiltration associated with 24-hour contact
hypersensitivity reactions. These data (mean ~ SEM)
are derived from the same mice whose ear thickness
values are shown in Figure 3. The reduction in
leukocyte infiltration observed in animals treated
with spiperone was significant when compared to the
reactions observed in animals treated with vehicle
alone (*p~0.05), while haloperidol, trazadone and
mianserin did not significan~ly suppress the leukocyte
infiltration associated wïth contact hypersensitivity.
` Fiqure 5 - Effect of spiperone applied topically
during the p2riod of sensitization on the tissue
swelling associated with oxazolone-induced contact
hypersensitivity reactions. Oxazolone was applied to
the abdomens of BALB/c mice on day 0. The change in
W093/12789 PCT~US92/11205
11 24~.2~7~
ear thickness was determined 24 ~ lrs aft~.- challenge
with oxazolone on day 6. Treatment with spiperone (50
~1 of 0.08% spiperone in propylene glycol) applied to
the abdomens on days -2, -1, 0, 1 and 2 significantly
diminished contact hypersensitivity reactions in the
right ears of the treated animals (**p<0.01 when
compared to the right ears in the control mice treated
with vehicle).
FiqurQ 6 - Effect of spiperone applie~ topically
lo du ~ the period of se~ itizaticn on the leukocyte
inJ -,ration associated with oxazolone-induced contact
hyp ~ensitivity reactions~ These data (mean + SEM)
re ~--;m the same mic~- hose ear thickness
ea ~ments are pres ~d in Figure 5. Topical
~reatment with spipe significantly diminished the
reactions when compa ~ those in vehicle-t~eated
mice (**p<O.Ol).
Fiqure 7a.b,c fect of topi~ lly administered
spiperone on tissue sw~lling ass~ciate~~ with
oxazolo~e-induced contact hyper- itivity re~_tions.
Oxazolone was ~- lied to both e ~f 1ll mice and the
change in ear ~. skness was mea~ ~ at a specified
interval thereafter. a. One hour after oxazolone
challenge, 4.0% spiperone in ethanol:propylene
glycol:olive oil was applied to both surfaces of the
right ears of some mice, whereas vehicle alone was
applied to both surfaces of the right ears of the
control (0% spiperon~) mice. The ears were measured
24 hours after oxazolone challenge. Local treatment
with 4% spiperone suppressed swelling in the treated
ear (**=p~Q.01 vs either contralateral oxazolone
treated ears or ears of vehicle treated group) a~
diminished the swelling in the contralateral ears
(**=p<C 1 vs left ears of vehicle treated group). b.
Two ho~rs before oxazolone challenge, 0.13% spiperone
in Vehicle-N was applied to ,both surfaces of the right
ears of some mice, whereas vehicle alone was applied
W093/12789 PCT/US92/11205
~12~
12
to both surfaces of the ears of the control (0
spiperone) animals. The ears were measured 24 hours
after oxazolone challenge. Local treatment of the
right ear with spiperone significantly suppressed
tissue swelling in the treated ear (**p<o.Ol vs
contralateral oxazolone treated ears or vs right ears
of vehicle treated group). c. Twenty-two hours after
oxazolone challenge, 0.13~ spiperone in Vehicle-N was
applied to both surfaces of the right ears of some
mice, whereas vehicle alone was applied to both
surfaces of the ears of control (0% spiperone) mice.
The change in ear thickness was determined 24 hours
after treatment with spiperone, i.e. at 46 hours after
~ challenge with oxazolone. Treatment with spiperone
lS significantly diminished contact hypersensiti~ity
reactions in the right ears of the treated animals
(*=p<O.Ol when compared to the right ears in the
control mice, and p<0.05 when compared to the
contralateral ears of the same mice). The reactions
in the left ears of the mice treated on the right ears
with spiperone were also reduced when compared to
reactions in the left ears of the vehicle-treated mice
(p<O. 01) .
Fiqure 8a,b,c - Effect of topical treatment with
spiperone on leukocyte infiltration associated with
oxazolone-induced contact hypersensitivity reactions.
These data (mean + SEM) are from the same mice whose
ear thickness measurements are presented in Figure
7a,b,c. Biopsies were performed 24 hours (a, b) or 46
hours (c) after application of oxazalone. Topical
treatment with spiperone significantly diminished the
reactions when compared to those in vehicle-treated
mice (**=p<O.Ol). In Figure 8a, the slight effect of
treatment of the right ears with spiperone on
reactions expressed in the left ears of the same mice
was not significant (p~0.05).
WO93/12789 PcT/us92/1l2nS
13 2 1 2 ~ 6 l 8
Fi~ure 9 - Effect of topically administered
spiperone on tissue swelling associated with DNFB-
induced contact hyperse~s-tivity reactions. DNFB was
applied to both ears of ~57BL/6J mice. One hour
later, 0.5% spiperone was applied to both surfaces of
the right ears of some mice, whereas vehicle alone was
applied to both surfaces of the right ears of the
control (o% ~piperone) mice. The change in ear
thickness was determined 24 hours after challenge with
DNF~. Treatment with spiperone significantly
diminished contact hypersensitivity reactions in the
right ears of the treated animals (**p<0.01 when
compared to the'right ears in the control mice, and
~. p<0.05 when compared to the contralateral ears of the
same mice). The reactions in the left ears of the
mice treated on the right ears with spiperone were~
also ~educed slightly when compared to reactions in
the left ears of the vehicle-treated mice (*p<0.05).
Fiaure 10 - Effect of topical treatment with
spiperone on leukocyte infiltration associated with
DNFB-induced contact hypersensitivity reactions.
These data (mean + SEM) are from the same mice whose
ear thickness measurements are presented in Figure 9.
Topical treatment with spiperone significantly
diminished the reactions when compared to those in
vehicle-treated mice (**p<0.01). The slight effect of
treatment of the right ears with spiperone on
reactions expressed in the left ears of the same mice
was not significant (p>0 ~5).
30 ~ Fiaure 11 - Comparative effects of varying doses
of systematically administered spiperone methyl
quaternary ammonium bromide salt versus spiperone on
tissue s~elling associated with oxazalone-induced
cutaneous contact hypersensitivity reactions.
Spiperone or spiperone methyl quaternary ammonium
bromide salt were administered to Balb/c mice 1 hour
after challenge for contact hypersensitivity. The
WO93/12789 PCT/US92/11205
2 ~ 2 ~ ~ r7 ~ 1 ~
change in ear~thickness (post-challenge value minus
baseline pre-challenge value) was measured 24 hours
after oxazalone challenge. The data is presented as
the mean + sEM. The reduction in ear swelling
observed with spiperone and spiperone methyl
quaternary ammonium bromide salt was significant when
compared to the reactions observed in the control,
vehicle treated animals at all dosage levels (30
mg/kg, 15 mg/kg, 6 mg/kg, and l.5 mg/kg, all given
intraperitoneally in 10% DMSO, 90~ water~.
Fiqure 12 - Comparative effects of systemic
treatment (intraperitoneal) with vehicle (o mg/kg) or
varying doses of spiperone or spiperone methyl
~-quaternary ammonium bromide salt on leukocyte
infiltration associated with 24-hour contact
hypersensitivity reactions. The data (mean ~ SEM)~are
derived from the same mice whose ear thickness values
are shown in Figure ll. The reduction in leukocyte
infiltration observed in animals treated with 30
mg/kg, l5 mglkg, 6 mg/kg and l.5 mg/kg of spiperone ;~
was significant when compared to the reactions
observed in animals treated with vehicle alone
(P<=0.05). Doses of 30 mg/kg, 15 mg/kg and 6 mg/kg of
spiperone methyl quaternary ammonium bromide salt
resulted in significant suppression of leukocyte
infiltration.
Detailed De~cription of the Invention
Definitions
The term alkyl, as used herein, unless otherwise
Ispecified, refers to a saturated straight, branched,
or cyclic hydrocarbon of Cl to C20, including methyl,
ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl,
pentyl, cyclopentyl, isopentyl, neopentyl, hexyl,
isohexyl, cyclohexyl, 3-methylpentyl, 2,2-
dimethylbutyl, and 2,3-dimethylbutyl.
WO93/12789 PCT/US92/11205
2~26~ ~
The ~ rm aryl, as used herein, and unless
otherwise specified, rffers to phenyl or substituted
phenyl, wherein the substituent is independently halo,
alkyl, or oxy(alkyl) (f~- example, ~ethyoxy, ethoxy,
S etc.), and wherein the -yl can have up to three
substituents.
I. Structure and Synthe~i~ of Spiperon~ Derivatives
The parent spiperone is ~-t'3-(p-
fluorobenzoyl)propyl~ phenyl-1,3,8-
lo triazaspiro[4.5~decan-4-one, which has the structure
illustrated below. ~`
o
C--
C61~5
As demon~trated in Example 1, the parent
spiperone has significant immunosuppressive activity.
However, uncomplexed or unmodified spiperone also has
significant neuroleptic effect when administered
systemically. However, spiperone can be complexed, or
chemically modified without undue experimentation
using methods known to those skilled in the art, to
`0 retain its immur ~uppressive activity and eliminate
the undesired neuroleptic effect ~y decreasing the
ability of ;he compound to bind to dopamine or
serotonin receptors. Alternatively, Example 1 shows
that spiperone can be given topically to produce local
limmunosuppression without inducing a significant
neuroleptic effect.
Compounds containing the spiperone nucleus, and
methods of synthesis thereof, are disclosed in V~S.
Patent Nos. 3,155,669; No. 3,155~670; No. 3,161,644;
and l~o. 3,238,216; all of which are hereby
incorporated by reference. The spiperone derivatives
W093/12789 PCT/US92/11205
2 ~ 16
disclosed herein can be made accordi~g to known
procedures, for example as disclosed in the patents
identified herein, or by obvious modifica~ions of
kncwn procedures.
The compounds ~ontaining the spiperone nucleus
can be complexed, or chemically modified, if
necessary, to re~ain immunosuppressive activity and
prevent the undesired neuroleptic effect.
Other compounds that contain the spiperone nucleus,
and which can be complexed or chemically modified if
necessary to prevent a neuroleptic effect for systemic
delivery, are of the formula:
o
~- . \\
R3
R4~N!~ /\~' ~R2
R
wherein:
R~ = H; alkyl, specifically including CH3-,
cyclohexyl, (CH3)2CH-, CH3(CH2)3-, (CH3)2CHCH2-~
CH3CH2CH(CH3)-, (CH3)3C-, and -CH3(CH2)p; Y-CH2(CH2) n~
or Ar~, specifically including C6Hs-,
(2, 3, or 4)-(OCH3)C6H4- and (2, 3, or 4)-(CH3)C6H4-;
2 0 2-X--C6H4~, 3--X-C6H4-, or 4--X-C6H4-;
R2 = H or C~ to C20 alkyl;
R3 = H; alkyl, specifically including -CH3,
CH3CH2-, CH3CH2CH2-, ( CH3)2CH-, or CH3 ( CH2) n~;
CN ~CH2) 2-; X~ ( CH2) n-; X- ( CH2) nCO~;
NH2C(NH)NHC(NH) (aryl) (CH2)o~; or X-(aryl)-(CH2)n-;
R4 = H, C6H5CH(CH2CH3)CH2-, C6HsCH(CH3) (CH2)2-,
C6HsCH2CH(CH3)CH2~, C6H5CH2CH2CH(CH3)-,
C6H5cH(cH3)(cH2)3 ~
(2, 3, or 4)-(alkyl)-C6H4CH(CH3)(CH2)3-,
.30 (2, 3, or 4)-(alkyloxy~-C6H4CH(CH3)(CH2)3,
WO9~/12789 PCT/US92/11205
17 21~2 & 57~
(2, 3, or 4)-X-C6H4-alkyl, specifically including
(2, 3, or 4)-X-C6H4CH(CH2CH3)CH2-, -
(2, 3, or 4)-X-C6H4C~(CH3)(CH2)-
4-X-C6H4CH(CH3)(CH2) 2- ~ and
4--X-C6H4-CH(CH3)(CH2)3-; C6H5CH(OCH3)tCH2)2-,
C6~sCH -IH-CH2-, C~5CO(CH2)3-, C6H5CO(CH2) 4 - ~
\CH2
(2, 3, or 4)-(alkyl)-C6H4CO(CH2)3~,
(2, 3, or 4)-(alkyl-oxy~-C6H4CO(CH2) 3-
(2, 3, or 4)-X-C6H4CO(CH2) n~
?-thienyl-C-(cH2)
Ar ~ H(CH2) n~
Arl
~ (2, 3, or 4)-X-C6H4C(CH3)CH~CH2) 2-, where the
conformation about the double bond is cis or trans,
(2, 3, or 4)-X-C6H4C(CH3)CHCH2-, where the conformation
about the double bond is cis or trans,
(2, 3, or 4 ? -X-C6H4COCH=CHCH2-,
Y-CH2(CH2)n-, Ar~-~CH2)D-, Cl to C20 alkyl, X-(CH23nCO-, or
X~tCH2)n~;
n = l to 6;
p is l to 20;
X = is independently F, Cl, Br, I, OCH3, S03- ~
NH2, H, -OH, -COOH, -COOR, -SO3H, -CN, -NHSO3H, -NO2, or
-S2N~2i
Y = H, F, Cl, Br, I, -SO3, -P04=~ -OH, -SH, -SCH3,
-CH3SO2, -NH2, or -CO2-; and
Ar~ is, independently, aryl, ~2, 3, or 4-X-C6H4-),
(2, 3, or 4)-(CH2X)C6H4-, (2, 3, or 4)-(CX3?C~H4-,
(2, 3, or 4)-(CHX2)C6H4~, 2-thienyl, or (2, 3, or 4)-X-
C6H4CH2-;
or its pharmaceut.cally acceptable s~lt,
including any quaternary salt known by ti. ~ in the
art, and specifically including the quaternary
ammonium salt of the formula -NR+Z-, wherein R is alkyl
(and in particular methyl or ethyl) or benzyl, and Z
is a counteranion, including chloride, bromide,
WO93/12789 PCT~US92J11205
2 ~ 2 ~
iodide, -O-alkyl, toluenesulfonate, methylsulfonate,
sulfonate, sulfate, phosphate, or carboxylate (such as
benzoate, succinate, acetate, glycolate ! propionate,
maleate, malate, citra~e, tartrate, ascorbate,
benzoate, cinnamoate, mandeloate, benzyloate, and
diphenylacetate).
Those forms of spiperone that are particularly
useful for systemic delivery are those in which R~
through ~ are chosen to minimize neuroleptic activity
'O and to maximize immunosuppressant activity of the
molecule, or wherein spiperone or the spiperone
derivative is in the form of a quaternary salt.
The potential utility of any one of the above-
~-~ described forms of spiperone to act as an
immunosuppressant can be conveniently determined by
synthesi7.ing the compound and testing it in the
biological assay described in Example 1. Also, the
neuroleptic activity of the spiperone derivative or
salt can be evaluated as described in Example 3.
The efficacy of spiperone derivatives can also
be assessed using animal models of allograft
rejection, experimental allergic encephalomyelitis,
lupus erythematosus, Freund's adjuvant arthritis
and/or graft versus host disease. Measurement of
their ability to bind to serotonin or dopamine
receptors can be carried out as described in detail in
Example 3, or by their lack of ability to act as a
tranquilizer or neuroleptic in mammals, for example,
by demonstrating that they are no different than
pl~cebo in the hot plate test of Eddy, et al., J.
! ` Pharmacol. 107:385 (1953) and 110:135 ~1954).
The chemically unrelated serotonin receptor
antagonists, trazadone and mianserin, and the dopamine
receptor antagonist, haloperidol, are not effective in
suppressing contact hypersensitivity. Qn this basis,
it i~ clear that the mechani~m of action of spiperone
and spiperone derivatives in suppressing the immune
WO93/127~9 PCT/~S92/11205
19 212~678
response is independent of their serotonin or dopamine
eceptor blocking properties. Therefore, spiperone
derivatives with immunosuppressive eff~ct yet without -~
neuroleptic effect can be provided for systemic
delivery by the me~hod of selec~ion disclosed
generally herein .
II . Complexation or Modif icatio~ of the SpiperoIle
Nucleu~ to Prevent Significant Neuroleptic Effect
As discussed above, immunosuppressive compounds
with a spiperone nucleus that have a neuroleptic ~:
effect can be complexed or modified to eliminate that
ef~ect for systemic delivery, by one or more of the
following processes.
A. Decreasing the Lipophilicity, equivalent to
lS Increasing the Rydrophilicity of the Co~pound
Compounds with a spiperone nucleus that exhibit
an immunosuppressive effect yet also exhibit a
neuroleptic effect can be modified to minimize the .
neuroleptic effect by decreasing the lipophilicity
(equivalent to increasing the hydrophilicity) of the ~-
molecule. This can be done by adding one or more
charged side chain(s) onto the molecule or by altering
the existing side chain to make it more polar. The ~;
hydrophilicity of spiperone derivatives will in
general increase when charged substituents are added.
For example:
N~
~NH3- CHz CH2- N ~ N
~3
WO93~12789 PCTJUS92/11205
,. 1 2 ~t 6 I~3
20 ~ :
would be expected to be much more hydrophilic than the
parent compound.
Moerlein et al. (Int. J. Nucl. Med._Biol.
12:353-356, 1985) have synthesized a number of forms
of spiperone designed to increase the lipophilicity of
the compound, and its potential ability to cross the
blood brain barrier, where they interact with dopamine
and serotonin receptors.
B. Increa~ing the 8ize of the Molecule
Another technique for reducing the central
nervous system (CNS) effects of compounds that
contains a spiperone nucleus is to increase the size
of the molecule via a covalent linkage to a large
~.~ moiety (e.g., albumin or polyethylene glycol), using
standard techniques of organic synthesis or by
choosing a spiperone derivative with large
substitutions (Rl, R2, R3, or R4).
C. Complexing the Compound with Spiperone Nucleus
with a Cyclic Molecule
A fourth method for reducing the central nervous
system (CNS) effects of a compound that contains a
spiperone nucleus includes forming a non-covalent
complex of the compound with a cyclic molecule such as
a cycloamylose (e.g., a cyclodextrin such as
B-cyclodextrin), which has a spatial arrangement of
hydroxyl groups whereby the outer surface of the ring
formed by the cycloamylose is hydrophilic and the
inner surface is lipophilic. When utilized in aqueous
solution, this structure permits molecules (or parts
thereof), termed "guest molecules", which are less
'polar thaln water and which are of suitable dimensions,
to be incorporated into the lipophilic inner cavity,
such that the cycloamylosetguest molecule complex
presents to the blood-brain barrier as a relatively
large and polar compound which is unable to penetrate
the barrier. Such complexes may be prepared by any
method known to the art, including those described in
WO93~12789 PCT/US92/1~205
21 2~ 26~8
u.s Patent No. 4,555,504, which discloses
B-cyclodextrin complexed with digoxin.
Spiperone altered or complexed by any of the
above methods (with the effect of reducing the CNS
effeGts of the compound to an acceptable level), and
which exhibits the ability to suppress an immune
response, is referred to herein as 'la spiperone
derivative without significant neuroleptic effect."
The efficacy of any such spiperone entity as an
immunosuppressant can be tested in the assay described
in Example 1 below. Whether the same entity is
capable of inducing the neuropharmacological side
effects observed for spiperone can be assayed by, for
~r example, the hot plate test of Eddy et al., J.
_armacol. 107:385 (1953) and 110:135 (1954), or by
the method of Example 3.
The central nervous system side effects of a
spiperone derivative can be estimated using molecular
modeling and/or pharmacophore analysis. The dopamine
and serotonin receptors are well characterized and
strategies for estimating binding of drugs to these
receptors are well established. For example, Schmidt,
et al., Molecular PharmacologY 38:511-516 (1990),
describe an algorithm for estimating the binding
2S affinity of drugs to the
5-HT receptor. Also, a composite pharmacophore
analysis and chemical database screening strategy is
de~cribed by Sleight, et al, Naunyn-Schmiedeberqs
Arch. Pharmacol. 343:109-116 (1991~, and Schmidt, A.W.
and Peroutka, S.J., Mol. Pharmacol. 36(4):505-511
'(19~9). ;R~ through R4 can be chosen to minimize
serotonin and/or dopamine receptor binding using these
or similar approaches and the derivatives can then be
tested f~r immunosuppressive activity, as described in
example 1.
WO93/12789 PCT/US92/1120s
.
D. Admini~tration as a Quaternary Salt
spiperone or its above-defined derivative can be
ad~inistered in the form of a pharmaceutically ~
accep~able quaternary salt. Quaternary salts are :
typically less lipophilic than the corresponding
unquaternized compound, and therefore have a decreased
effect on the central nervous system. Nonlimiting
examples of quaternary salts that can be used include,
but are not limited to salts prepared from methyl
lo chloride, methyl bromide, methyl iodide, methyl
sulfate, ethyl sulfate, methyl benzene-sulfonate,
methyl p-toluenesulfonate, et~yl p-toluenesulfonate,
ethyl chloride, ethyl bromide, ethyl iodide, n-propyl
~ chloride, n-propyl bromide, n-butyl bromide, isobutyl
bromide, sec-butyl bromide, n-amyl bromide, n-hexyl
chloride, benzyl chloride, benzyl bromide, and ethyl
sulfate.
More generally, spiperone or its derivative can
be administered as a quaternary salt of the formula
-NR+Z~, wherein R is alkyl (and in particular methyl or
ethyl) or benzyl, and Z is a counteranion, including
chloride, bromide, iodide, -0-alkyl, toluenesulfonate,
methylsulfonate, sulfonate, sulfate, phosphate, or
carboxylate (such as benzoate, succinate, acetate,
glycolate, propionate, maleate, malate, citrate
tartrate, ascorbate, benzoate, cinnamoate, mandeloate,
benzyloate, and diphenylacetate).
ITI. Therapeutic Compo~itions
Mammals, and specifically humans, suffering from
, 30 Ipathogenic immune responses can be treated by topical
or systemic administration to the patient of an
effective amount of spiperone or its derivative or
pharmaceutically acceptab~e salt, optionally in the
presence of a pharmaceutically acceptable carrier or
diluent.
W093/127~9 PCT/US92/11205
~ 212~7~
The spiperone derivative is administered
subcutaneously, intravenously, intraperitoneally,
intramuscularly, parenterally, orally, submucosally,
by inhalation, transdermally via a slow release patch,
S or topically, in an effective dosage range to cause
immunosuppression. Typical systemic dosages for all
of the herein described conditions are those ranging
from O.l mg/kg to 500 mg/kg of body weight per day as
a single daily dose or divided daily doses. Typical
dosages for topical application are those ranging from
O.OOl to 100% by weight of the active compound. In
general, local immunosuppression can be achieved by
administering topically lower doses of spiperone
~ derivatives than would be required if ~he agents were
administered systemically. The effective dosage of
the parent compound, spiperone, for systemic
immunosuppression is believed to be higher than the
efrective dosage of spiperone for inducing a
neuroleptic effect.
The spiperone derivative is administered for a
s~ficient time period to alleviate the undesired
s~ toms and the clinical signs associated with the
condition being treated.
The active compound is included in ~he
pharmaceutically acceptable carrier or di~uent in an
amount sufficient to deliver to a patier
therapeutic amount of compound of the spi~erone
derivative in vivo in the absence of serious toxic
effects.
The concentration of active compound in the drug
composition will depend on absorption, inactivation,
and excretion rates of the drug a~ well as other
factors known to those of skill in the art. It is to
be noted that dosage ~lues will also vary with the
severity of the condi~ion to be alleviated. It is to
be further understood that for any particular subject,
specific dosage regimens should be adjusted over time
WO93/12789 PCT/US92/11205
2 ~ 2 ~
24
according to the individual need and the professional
judgment of the person administering or supervising
the administration of the compositions, and that the
dosage ranges set forth herein are exemplary only and
are not intended to limit the scope or practice of the
claimed composition. The active ingredient may be
administered at once, or may be divided into a number
of smaller doses to be administered at varying
intervals of time.
A preferred mode of administration of the active
compound for systemic delivery is oral. Oral
compositions wilI generally include an inert diluent
or an edible carrier. They may be enclosed in gelatin
~ capsules or compressed into tablets. For the purpose
of oral therapeutic administration, the active
compound can be incorporated with excipients and used
in the form of tablets, troches, or capsules~
Pharmaceutically compatible binding agents, and/or
adjuvant materials can be included as part of the
composition.
The tablets, pills, capsules, troches and the
like can contain any of the following ingredients, or
compounds of a similar nature: a binder such as
microcrystalline cellulose, gum tragacanth or gelatin;
an excipient such as starch or lactose, a
disintegrating agent such as alginic acid, Primogel,
or corn starch; a lubricant such as magnesium stearate
or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or
saccharin; or a flavoring agent such as peppermint,
methyl sa~licylate, or orange flavoring.
When the dosage unit form is a capsule, it can
contain, in addition to material of the above type, a
liquid carrier such as a fatty oil. In addition,
dosage unit forms can contain various other materials
which modify the physical form of the dosage unit, for
WO93/12789 PCT/US92/11205
25 ~ 7~ -
example, coatings of sugar, shell -, or other enteric
agents .
The spiperone deriv ive or its salts can be
administered as a component of an elixir, suspension,
syrup, wafer, chewing gum or the like. A syrup may
contain, in addition to the active compounds, sucr~se-
as a sweetening agent and certain preservatives, dy~s
and colorings and flavors.
The spiperone derivative can also he mixed with
other active materials which do not impair the desir 1
action, or with materials that sup~ ement the desired
action, such as antibiotics, antif~ngal~,
antiinflammatories, antivirals, or other
immunosuppressive agents~
Solutions or suspensions used for parenteral,
intradermal, subcutaneous, or topical application can
include the following components: a sterile diluent
such as water for injection, saline solution, fixed
oils, polyethylene glycols, glycerine, propylene
glycol or other synthetic solvents; antibacterial
agents such as benzyl alcohol or methyl parabens;
antioxidants such as ascorbic acid or sodium
bisulfite; chelatin~ agents such as
ethylenediaminetetr~ Icetic acid; buffers such as
' 25 acetates, citrates ~r phosphates and agents for the
adjustment of tonicity such as sodium chloride or
dextrose. pH can be adjusted with acids or bases,
such as hydrochloric acid or sodium hydroxide. The
parenteral preparation can be enclosed in ar~Qules,
disposable syringes or multiple dose vials made of
glass or plastic.
If administered intraveno~ ly, preferred
~arriers are physiological saline,bacteriostati
water, Cremophor ELTM (BASF, Parsippany, NJ) or
phosphate ~-~ffered saline (PBS).
In a preferred embodiment, the active compounds
are prepared with carriers that will protect the
WO93/12789 PCT/US92/11205
7~ 26
compound against rapid elimination from the body, ~uch
as a controlled release fo~mulation, including
implants and microencapsulated delivery systems.
Biodegradable, biocompatible polymers can be used,
such as ethylene vinyl acetate, polyanhydrides,
polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such
formulations will be apparent to those skilled in the
art. The materials can also be obtained commercially
from Alza Corporation and Nova Pharmaceuticals, Inc~
Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies
to viral antigens) are also preferred as
~-pharmaceutically acceptable carriers. These may be
prepared according to methods known to those skilled
in the art, for example, as described in U.S. Patent
No. 4,522,811 (which is incorporated herein by
reference in its entirety). For example, liposome
formulations may be prepared by dissolving appropriate
lipid(s) (such as stearoyl phosphatidyl ethanolamine,
stearoyl phosphatidyl choline, arachadoyl phosphatidyl
choline, and cholesterol) in an inorganic solvent that
is then evaporated, leaving behind a thin film of
dried lipid on the surface of the container. An
aqueous solution of the spiperone derivative is then
introduced into the container. The container is then
swirled by hand to free lipid material from the sides
of the container and to disperse lipid aggregates,
thereby forming the liposomal suspension.
Suitable vehicles or carriers for topical
application can be prepared~by conventional
techniques, such as lotions, suspensions, ointments,
creams, gels, tinctures, sprays, powders, pastes,
slow-release transdermal patches, suppositories for
application to rectal, vaginal, nasal or oral mucosa.
In addition to the other materials listed above for
systemic administration, thickening aqents,
WO93/1278~ PCT/US92~11205
` 27 2126~7~
emollients, and stabilizers ca~ ~e used to prepare
topical compositions. Jxamples of thickening agents
include petrolatum, beeswax, xanthan gum, or
polyethylene, humectants such as sorbitol, emollients
such as mineral oil, lanolin and its derivatives, or
squalene. A number of solutions and ointments are -
commercially available, especially for ophthalmic
applications.
Spiperone derivatives can be provided in the
form of pharmaceutically-acceptable salts. As used
herein, the term "pharmaceutically-acceptable salts or
complexes^' refers to salts or complexes that retain
the desired biological activity of the parent compound
~ and exhibit minimal, if any, undesired toxîcological
lS effects. Examples of such salts are (a) acid addition
salts formed with inorganic acids (for example, ~;
hydrochloric acid, hydrobromic acid, sulfuric acid,
phosphoric acid, nitric acid, and the like), and salts
formed with organic acids such as acetic acid, oxalic
acid, tartaric acid, succinic acid, malic acid,
ascorbic acid, benzoic acid, tannic acid, pamoic acid,
alginic acid, polyglutamic acid, naphthalenesulfonic
acids, naphthalenedisulfonic acids, and
polygalacturonic acid; (b) base addition salts formed
with polyvalent metal cations such as zinc, calcium,
bismuth, barium, magnesium, aluminum, copper, cobalt,
niskel, cadmium, and the li~ or with an organic
cation formed from N,N-dibenzylethylene-diamine or
ethylenediamine; or (c) combinations of (a) and (b);
e.g., a zinc tannate salt or the like. Also, as
described in detail above, quaternary salts of the~ ~
active compounds cn also be administered. ;
WO93/12789 P~T/US92/11205 ;~
2 1 2 6 ~ 28
IV~ Immunosuppre~sa~t Activity of spiperone
Derivatives
Spiperone derivatives are capable of acting
systemically or ~opically to suppress the immune
response in animals. As such, the compounds, or
therapeutic compositions thereof, are useful for the
treatment of a myriad of i~munological disorders.
Examples of such disorders that are usually treated
systemically include those related to host rejection
~0 of foreign organ or tissue transplants; graft-vs-host
disease; and autoimmune diseases, such as rheumatoid
arthritis, juvenile rheumatoid arthritis, psoriatic
arthritis, psoriasis, autoimmune uveitis, multiple
~- sclerosis, allergic encephalomyelitis, systemic lupus
erythematosis, acute necrotizing hemorrhagic
encephalopathy, idiopathic bilateral progressive
sensorineural hearing loss, aplastic anemia, pure red
cell anemia, idiopathic thrombocytopenia,
polychondritis, scleroderma, Wegener's granulomatosis,
chronic active hepatitis, myasthenia gravis, atopic
dermatitis, Stevens-Johnson syndrome, idiopathic
sprue, Crohn's disease, Graves ophthalmopathy,
sarcoidosis, primary biliary cirrhosis, primary
juvenile diabetes, uveitis posterior, and interstitial
2S lung fibrosis; and allergic reactions, includin~
atopic dermatitis and contact dermatitis. Examples of
other immune disorders of the skin, mucosa, or eye
that generally are treated topically (although many
can also be treated systemically) include alopecia
areata, arthropod bites, lichen planus, cutaneous
, ~ ' lupus erythematosus, scleroderma, dry eye associated
with Sjogren's syndrome, and drug reactions.
The compounds are specifically useful in the
treatment of allograft re3ection, for example, of
~5 heart, kidney, and lung tissue, and in the treatment
of graft vs. hos~ disease associated with bone marrow
transplants.
WO93/12789 PCT/US92/11205 ~
29 2 ~ 2 ~ ~ 7 8
The ability of the neuroleptic agent spiperone
(8-t3-{p-fluorobenzoyl}propy~ phenyl-l~3~8-
triazaspiro-[4.s~decan-4-one) to inf luence the tissue
swelling and leukocyte infiltration associated with
contact hypersensitivity reactions in mice was
evaluated as described in detail in Example l. The
parent spiperone compound was used for the procedure
in Example l as a model of an active
immunosuppressant. Other compounds with a spiperone
nucleus can be measured against this model, and are
considered active if they suppress the leukocyte
infiltrate and/or the swelling response by at least
40% 24 hours after specif`ic antigen challenge.
~ In the procedure of Example l, contact
hypersensitivity reactions were elicited by applying
the haptens oxazolone or dinitrofluorobenzene
topically to one or both ears five to eight days after
epicutaneous sensitization. When spiperone was given
subcutaneously at a dose of 150 mg/kg, l hour after
challenge with oxazolone, cutaneous contact
hypersensitivity to this hapten was almost totally
abrogated. A dose of 40 or 30 mg/kg subcutaneously
also significantly suppressed the reactions but to a
lesser degree than the higher dose. When applied
topically, preparations of spiperone significantly
suppressed both the tissue swelling and the leukocyte
infiltratior associated with the elicitation phase of
contact hypPrsensitivity to either oxazolone or
dinitrofluorobenæene. Topical treatment with
spiperone also suppressed the sensitization phase of
contact sensitivity. However, mice treated topically
with spiperone, unlike those treated systemically,
exhibited no drowsiness or other evidence of central
nervous system effects.
Spiperone expresses both serotonin and dopamine
receptor antagonist activity. However, unlike
spiperone, it was discovered that the chemically
WO93~12789 PCT/US92/11205
2 12 !~
unrelated serotonin antagonists, trazadone and
mianserin, and the dopamine receptor antagonist,
haloperidol, were not effective in suppressing contact
hypersensitivity. Additionally, the methyl quaternary
S ammonium bromide salt of spiperone has substantially
reduced CNS activity, while retaining
immunosuppressive activity. On the basis of this, it
is clear that the mechanism of action of spiperone on
the immune response is independent of its serotonin or
dopamine receptor blocking properties, and therefore,
spiperone derivatives with immunosuppressive effect
yet without neuroleptic effect can be provided by the
method of selection disclosed generally herein.
~ Example l: Inhibition of Induced Contact
Hypersensitivity.
Six-to-8-week-old female C57BL/6J or BALB/c mice
were obtained from the Jackson Laboratory, Bar Harbor,
Maine or from Charles River Laboratories, Kingston
Facility, Stoneridge, NY, respectively.
Spiperone, mianserin, trazadone, haloperidol and
oxazolone were purchased from the Sigma Chemical Co.
(St. Louis, MO).
Oxazolone-Induced Contact Hypersensitivity -
Sensitization and challenge for contact
hypersensitivity were performed as follows. The
abdomens of the mice were shaved with electric
clippers, 50 ~l of a 4% (w/w) solution of oxazolone in
4:1 (v:v) acetone:olive oil were applied to the shaved
abdomen, and 5 ~l of the same solution were applied to
each hind footpad. ~ive to eight days later, the mice
were challenged for contact hypersensitivity by
applying l0 ~I of a 0.5% (w:w) solution of oxazolone
in 4:l (v:v) acetone:oliYe oil to both the inner and
outer surface of the right ear of each mouse (in the
case of mice treated systemically with spiperone) or
to both ears (in the case of mice treated topically
with spiperone).
WOg3/1278~ PCT/US92/11205
31 21 2g~ 6
Dinitrofluorobenzene-Induced contact
HYpersensitivity - Mice were treated in an identical
manner as above~ except that 0.2% (v:v) l-fluoro-2,4-
dinitrobenzene (DNFB) in acetone was used for both
sensitization and elicitation of the contact
hypersensitivity response.
systemic spiperone Treatment - One hour after
the application of oxazolone for elicitation of
contact hypersensitivity, mice were treated
subcutaneously with spiperone (150 or 30 mg/kg body
weight) in 0.l ml of carrie~ (Cremophor EL, BASF,
Parsippany, NJ), or with 0.i ml of carrier alone. In
a separate experiment, mice were treated in a similar -
~ fashion with ~ mg/kg body weight of trazadone,
mia,tser ll, ha_operidol, or spiperone in 0.l ml ol~-~
oil or with olive oil alone.
Topical Spiperone Treatment - To test whether
~,iperone affe~t:ed the sensitization phase of contact
hypersensitiv~ , 50 ~l of 0.08% spiperone in
propylene gly~ 1 was applied to the shaved abdomens of
the mice on days -2, -l, 0, l and 2, with the day of
oxazalone sensitization being designated day 0. To
test the effects of spiperone on the expression of
contact hypersensitivity in mice already sensitized to
oxazolone; mice were treated with spiperone topically
at two hours before or one or twenty-two hours after
challenge for contact hypersensitivity, by applying l0
~l of a solution of spiperone in vehicle to both sides
of the right ear. In the case of oxazolone-sensitized
mice treated one ~our after challenge, a 4~ (w/w)
spiperone suspenslon in 4:l:5 absolute
ethanol-propylene glycol:olive oil was used, while
0.13~ (w/w) spiperone solution in Vehicle-N
(Nel:~rogena Corp., Los Angeles, CA) was used at the
oth r time points. In the case of the DNFB-sensitized
mice, 0.5% ~w/w) spiperone in absolute ethanol was
used.
W093/12789 PCT/USs~/l1205
~2~ 32
Evaluation of Ear swellinq Response - -
Immediately before and 24 or 46 hours after
application of oxaæolone or DNFB, ear thicknesses were
determined with an engineer's micrometer. The
increment (delta~ in ear thickness (ear swelling) was
calculated as the 24- or 46-hour value minus the
baseline (pre-challenge) value and expressed in units
of l0-~Nm. Mice were killed by cervical dislocation
after the measurement of 24-hour ear thickness was
obtained, and the ears were processed for histologic
examination.
Ouant_fication of Leukocyte Infiltration - In
most experiments, both ears of each mouse were fixed
~ in 4.0% buffered formalin and then processed routinely
lS and embedded in paraffin for preparation of 6-7 ~m
thick hematoxylin and eosin-stained sections. In some
experiments (Figs. 2 and l0), ears were fixed and
processed into l ~ m thick, Epon-embedded, Giemsa-
stained sections. All of the sections were coded and
examined with an ocular grid at 400x under light
microscopy by an observer unaware of the identity of
the individual slides. The number of leukocytes/mm2 of
dermis was calculated by counting all of the leukocyte
cells in an area of at least 0.14 mm2 of dermis.
Statistical Analysis - Differences between
groups were assessed by the 2-tailed Student's t test
(paired for comparisons of left and right ears in the
same mice, unpaired for comparisons between different
groups of mice).
Results.
Effects of Systemic Treatment with Spiperone on
Expression_of Contac~ Hypersensitivity - ~he
subcutaneous administration of spiperone at a dose of
150 mg/kg, l hour after challenge for contact
hypersensitivity to oxazolone, markedly diminished (by
80~) the tissue swelling which developed in
association with the contact hypersensitivity response
WO93/12789 PCT/US92/11205
~.
2 ~ r~J 5~
(Fig. l). Figure 2 shows that the leukocyte
infiltration associated with the response in mice
treated with ~50 mg/kg spiperone was also diminished
by approximately the same amount ( f 1% reduction
compared to responses in mice not ~reated with the
drug~. However, at this dose, spiperone also produced
other remarkable systemic effects. The mice rapidly
became lethargic after administration of the drug,
and, by 23 hours after spiperone injection, the mice
exhibited profound depression of central nervous
system functiGa~ They appeared to be in a deep sleep,
neither ate nor drank, and responded weakly or not at
all to touch. They did, however, exhibit
~-~ responsivenes~ to pinch~
Some mice were treated with spiperone at 30
mg/kg subcutaneously (Figs. l and 2). At this dose,
spiperone diminished the tissue swelling associated
with contact hypersensitîvity to oxazolone to almost
the same extent as did the higher dose (68% reduction
with 30 mg/kg versus 80% reduction with 150 mg/kg) but
reduced the leukocyte infiltration associated with the
reaction by only 37% (Fig. 2). However, the central
nervous system e~fects of spiperone at 30 mg/kg were
substantially less pronounced that those observed at
the higher dose. Thus, the mice treated with
spiperone at 3Q mg/kg were less sleepy than those
treated with 150 mg/kg. However, the mice treated
with 30 mg/kg appeared somewhat l~thargic and were
less interested in food and water than were control
mice treated with carrier alone.
Systemic SpiPerone Versus Other Serotonin or
Dopamine Receptor Antagonists - In these experiments,
systemic spiper ne was compared to the serotonin
recept~r antagonists, trazadone or mianserin, and to
the dopamine receptor antagonist, haloperidol, for
their ability to inhibit cutaneous contact
hypersensitivity. At a dose of 40 mg/kg, only
W093/12789 PCT/US92/11205
2 1 2 6 li I ~ 34
systemic spiperone significantly reduced cutaneous
contact hypersensitivity (Fig. 3, 4). The degree of
lethargy in mice treated with 40 mg/kg of spiperone,
trazadone, mianserin or haloperidol systemically ~Fig.
l and 2), appeared to be about the same as that in the
mice treated with 30 mg/kg of spiperone systemically.
Systemic Spiperone versus the Methvl Ouaternary
Ammonium Bromide Salt_of Spiperone - In these -:
experiments, systemically administered spiperone and
systemically administered methyl quaternary ammonium .-
bromide salt of spiperone were evaluated for their
ability to inhibit cutaneous contact hypersensitivity.
~-~ At systemic doses of 30 mg/kg, 15 mg/kg, 6 mg/kg and
1.5 mg/kg the spiperone methyl quaternary ammonium
bromide salt produced significant suppression of the
tissue swelling associated with oxazalone induced
contact hypersensitivity, but with substantially less
effect on the central nervous system (CNS) (Figure ll)
than spiperone. Similar results were obtained when
the immune response was quantituted based on leukocyte
infiltration, with doses of lS mg/kg, 6 mg/kg, and 1.5
mg/kg of spiperone methyl quaternary ammonium bromide
salt producing substantial suppression of the
response, but with less CNS effects compared to
spiperone given at the same dose (Figure 12).
Effects of Spiperone on the Sensitization Phase of
Contact HyE~ersensitivity - For these experiment~, mice
were treated topically with spiperone in Vehicle-N or
Vehicle-N alone, applied to the abdomen beginning two
idays prîo~r to sensitization and continuing for a total
of 5 days (Figs. 5 and 6). Mice treated with
spiperone exhibited 64~ less tissue swelling and 70
less leukocyte infiltration at sites of hapten
challenge than did vehicle-treated mice (p<O.Ol for
either comparison). These data show that treatment
with topical spiperone can effectively inhibit the
WO93/12789 PCT/US92/11205
35 2 ~ 7 ~ :
sensitization phase of cutaneous contact
hypersensitivity.
Effects of Topical Spiperone on ExPression of Contact
Hypersensitivity - For these experiments, both ears of
each mouse were challenged for elicitation of contact
hypersensitivity by the application of oxazolone or
DNFB (as appropriate) to both surfaces of both ears~
TW~ hours before, one hour after or twenty-two hours
af ~r application of hapten, the right ears of some
mi~C were treated with spiperone in vehicle, applied
epicutaneously to both surfaces. The right ears of
control mice were similarly treated, but with vehicle
alone. Topical administration of a 4.0% suspension of
~ spiperone in absolute ethanol, propyle~e glycol, and
'5 olive oil one hour after hapten challenge resulted in
a marked diminution of the tissue swelling associated -
with contact hypersensitivity reactions elicited in
the right (spiperone-treated) ear and had a smaller,
but nonetheless significant, effect on the swelling
associated with the contact hypersensitivity reaction ~
elicited on the contralateral (untreated) ear (Fig. ~`
7a). Thus, reactions in the untreated right ears were
90 smaller than reactions in the right ears of
vehicle-treated mice, whereas reactions in the left
ears of mice treated on the right ears with spiperone
were reduced by 60% compared to the reactions in the
right ears of the vehicle-treated mice (Fig. 7a).
When the effect on lt~kocyte infiltration associated
with the contact hypersensitivity reactions was
assessed (Fig. 8a), the results were similar.
'Reactions in the spiperone-treated right ears were
diminished by 76% compared to the right ears of
vehicle-treated mice, whereas reactions in the left
ears of mice treated on the right ears with spiperone
were reduced only 22% compared to those in the left
ears of vehicle-treated mice.
WO93/127~9 PCT/US92/1~05
2~ ~S 7~ 36
A lower concentration of spiperone (0.013~),
applied topically to the right ear 2 hours before
(Fig. 7b and sb) or 22 hours after (Figs. 7c and 8c)
hapten challenge was also tested. The results
demonstrate that the lower concentration of spiperone
inhibited the majority of the tissue swelling and
leukocyte infiltration associated with contact
hypersensitivity reactions elicited at the site of
treatment (the right ear), but had no significant
effect on the intensity of the reactions elicited by
the same dose of hapten applied to the contralateral
(left) ear. Note that treatment with-either vehicle
had little or no effect on the responses (Figs. 7 and
~ 8).
Although topical application of spiperone was
extremely effective in diminishing both the tissue
swelling and the leukocyte infiltra~ion associated
with contact hypersensitivity reactions, these effects
were observed in the absence of detectable alterations ;
in the behavior of the mice. In contrast to mice
treated systemically with spiperone, the mice treated
topically with this agent appeared active and retained
apparently normal interest in food and water.
To evaluate the effect of topical treatment with
spiperone on contact hypersensitivity reactions
elicited with a different hapten, the effect of
topical treatment with a 0.5% suspension of spiperone
on the contact hypersensitivity reactions elicited
with DNFB was examined. Topical treatment with
spiperone significantly diminished the tissue swelling
associated with reactions to DNFB (by 45%, Fig. 9)~and
had an even more significant effect on leukocyte
infiltration (a reduction of 71~ compared to right
ears of vehicle-treated mice, Fig. 10). At this dose
of spiperone and with this hapten, the effect of
spiperone on reactions elicited in the left ears of
mice treated on the right ears with the drug were
W093/12789 PCT/US92/11205
. 2 L2~jr~
modest (28% reduction in tissue swelling and 18%
reduction in leukocyte infiltration compared to values
for the left ears of vehicle-treated mice, Fig. 9 and -~
lO). In fact, in this experiment, the effect of
S spiperone applied to ~he right ears on the leukocyte
infiltration associated with reactions elicited in the
left ears was not significant (p >0.05).
Example 2: Compariso~ of Immunosuppr~ssant versu~
Anti-inflammatory activity.
Mice were sensitized to oxazolone as described
in example l. Three days later, slow release ;
indomethacin pellets (0.05 mg, 3 week release) were
implanted subcutaneously under light ether anesthesia.
~.The dose of indomethacin delivered by these pellets
has been previously shown to completely block
prostaglandin synthesis in mice, by Jun, D.D., et al.,
J. Invest. Dermatol. 90:3ll (1988).
Three days later, mice were challenged for
contact hypersensitivity as in example l. When the
hypersensitivity response was assessed 24 hours later,
indomethacin was sho~n to have no c ~nificant effect
on the res~onse. These data show .ha~ a classic anti-
inflammatory agent, indomethacin, c~nlnot suppress the
immunologically specific oxazolone induced contact
hypersensitivity response.
Example 3: Evaluation of Serotonin Receptor Binding
Activity or Dopamine Receptor Binding
Activity of Spiperone Derivatives.
Spiperone derivatives which lack serotonin
receptor binding or dopamine receptor binding activity
can be identified as follows. A radiolabeled ligand
known to bind serotonin and/or dopamine receptors can
be bound to an appropriate substrate expressing one or
both of these'receptors. For example, radiolabeled
quipazine which is available commercia~ can be used
as the ligand. The spiperone derivative to be tested
is then incubated with the radiolabeled quipazine
ligand combination. Displacement of radiolabeled
WO93/12789 PCT/US92/11205
2126~ 1~ 38
ligand is positive evidence that the spiperone
derivative being tested can bind serotonin and/or
dopamine receptors. The amount of radiolabeled ligand
which is displaced is determined by an appropriate
standard curve which can also provide information
concerning binding affinities. The displaced
radiolabeled ligand can be quantitated using a
standard scintillation counter.
A detailed description of how to perform the ;
binding studies using 3H-quipazine and the example
follows:
Binding studies using 3H-quipazine are described
in detail by Milburn, C.M. and Peroutka, S.J., J.
~ Neurochem. 52:1787-1792 (1989). Briefly, rat cortices
are homogenized in 20 volumes of 50 mM Tris HCl buffer
pH 7.7 at 25~C and centrifuged at 49,000 x g for 10
min. The pellet is resuspended in fresh buffer and
incubated at 37C for 10 min. After the final
centrifugation, the pellet is resuspended in 80
volumes of Krebs-HEPES buffer (25 mM HEPES, 118 mM
NaCl, S mM KCl, 2.5 mM CaCl2, and 1.2 mM MgCl2 pH
adjusted to 7.4). Tissue (10 mg of original wet
weight) is added to assay tubes containing 0.8 nM
~3H~quipazine and displacing drug or buffer in a final
volume of 1 ml. Non-specific binding is defined using
1 micromole zacopride. After a 30 min incubation at
room temperature, the tissue is rapidly filtered under
vacuum through No. 32 glass fiber filters and rinsed
twice with S ml of 50 mM Tris~HCl buffer pH 7.7.
Radioactivity is quantified by liquid scintillation
counting.i~ All experiments~are performed three to six
times, each in triplicate; This same approach can be
used with other radiolabeled ligands such as
zacopride, granisetron, haloperidol, mianserin,
ketanserin, 5-HT, dopamine, droperidol, or ritanserin.
Spiperone derivatives which have binding
affinities for dopamine and/or serotonin receptors of
WO93~12789 ` PCT/US92/l1205
2 t 2 ~ ~ f ~3 :one/tenth or less than native spiperone are considered -
to be potentially useful as systemic
immunosuppressants if they are at least 50% as active
as native spiperone on a weight basis in suppressing
immunologically specific responses such as contact
hypersensitivity.
Example 4 Immunosuppressive and CNS Effect of the
Methyl Quaternary ~mmonium Salt of
Spiperone
The methyl quaternary ammonium bromide salt of
spiperone was prepared by the following procedure.
Spiperone (Sigma, 4.0 gm, lo mmol) was dissolved in a
1:1 mixture of warm methylene chloride and methanol
~. (80 mL). The solution was transferred to a Wheaton
pressure bottle. Methyl bromide (2N in ether, 8 mL,
16 mmol) was added~ The reaction vessel was heated in
an oil bath at 60C overnight. The reaction solution
was cooled and the white precipitate filtered, washed
with methanol, and dried at high vacuum at room
temperat~re. The pr luct was obtained as a white
solid (1.7 gm). Meltlng point, 245-246.5 C;
elemental analysis (-1.25 H20): calc. C, 56.20; H,
6.19; N, 8.19; Br, 15.58. Found: C, 56.13; H, 6.14;
N, 8.23; ~r, 15.65.
Figures 11 and 12 illustrate the effect of
spiperone and the methyl quaternary ammonium ~romide
salt of spiperone when injected intraperitoneally at
different dosages on oxazolone induced contact
hypersensitivity. The mice were injected with the
test compound one hour after oxazolone challenge, and
ear thickness measured at 24 hours after challenge.
The dosages used are indicated in Table 1. The CNS
effects were assessed by periodically observing the
activity of the animals over a 23 hour period
following injection of the test substance. The
following scoring system was used and corresponded to
the maximal effect observed during any observation
period.
WO93/12789 PCTJUS92/11205 -~
2 1 ~ ~ ~ 7 ~
.
4+ comatose
3+ Sleeping, but arousable
2+ Lethargic
l+ Less active than control
s o Normal
Table l :
CNS Effect of Methyl Quaternary Ammonium Salt of
Spiperone
lOGroup Dose (mg/kg) SPIP QUAT
. ';
A 30 +++ ++ ;~
B l5 +++ + .
C 6 ++ none
D l.5 ++ none
Control 0 none none
'"
As indicated in Figures ll and 12, the methyl
quaternary ammonium bromide salt of spiperone
significantly reduced ear swelling as compared to the
control at dosages as low as l.5 mg/kg, and had no
appreciable CNS effect at dosages up to 6 mg/kg.
Modifications and variations of the present
invention will be obvious to those skilled in the art ;
from the foregoing detailed description of the
invention. Such modifications and variations are
intended to come within the scope of the appended
. ~claims. , ~ ~
