CA2126678A1 - Use of spiperone or spiperone derivatives as immunosuppressant agents - Google Patents

Use of spiperone or spiperone derivatives as immunosuppressant agents

Info

Publication number
CA2126678A1
CA2126678A1 CA002126678A CA2126678A CA2126678A1 CA 2126678 A1 CA2126678 A1 CA 2126678A1 CA 002126678 A CA002126678 A CA 002126678A CA 2126678 A CA2126678 A CA 2126678A CA 2126678 A1 CA2126678 A1 CA 2126678A1
Authority
CA
Canada
Prior art keywords
spiperone
alkyl
bromide
group
derivative
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
CA002126678A
Other languages
French (fr)
Inventor
Richard J. Sharpe
Kenneth A. Arndt
Stephen J. Galli
Peter C. Meltzer
Raj K. Razdan
Howard P. Sard
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Beth Israel Deaconess Medical Center Inc
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US07/815,283 external-priority patent/US5290783A/en
Priority claimed from US07/831,429 external-priority patent/US5244902A/en
Priority claimed from US07/893,534 external-priority patent/US5574041A/en
Priority claimed from US07/893,536 external-priority patent/US5703088A/en
Application filed by Individual filed Critical Individual
Publication of CA2126678A1 publication Critical patent/CA2126678A1/en
Abandoned legal-status Critical Current

Links

Classifications

    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00Drugs for immunological or allergic disorders
    • A61P37/02Immunomodulators
    • A61P37/06Immunosuppressants, e.g. drugs for graft rejection

Landscapes

  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Engineering & Computer Science (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Transplantation (AREA)
  • Epidemiology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

A method for suppressing an immune response in a mammal by treating the mammal topically or systemically with an effective amount of spiperone or a spiperone derivative, or its pharmaceutically acceptable salt, including a quaternary salt.

Description

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 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 ~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 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.

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 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 ~- . \\

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 2 ~ 2 ~

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 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 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 ~.
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 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 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 . 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 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. , ~ ~

Claims (35)

We claim:
1. A method for treating a mammal in need of immunosuppression, comprising topically or systemically administering to the mammal an effective amount of a spiperone derivative of the formula:

wherein:
R1 = H; alkyl, Y-CH2(CH2)n-or Ar1, R2 = H or C1 to C20 alkyl;
R3 = H; alkyl, CN(CH2)2-; X-(CH2)n-; X-(CH2)nCO-;
NHC2(NH)NHC(NH)(aryl)(CH2)n-; or X-(aryl)-(CH2)n-;
R4 = H, C6HsCH(CH2CH3)CH2-, C6H5CH(CH3)(CH2)2-, C6H5CH2CH(CH3)CH2-, C6H5CH2CH2CHCH3-, C6H5CH(CH3)(CH2)3-(2, 3, or 4)-(alkyl)-C6H4CH(CH3)(CH2)3-, (2, 3, or 4)-(alkyloxy)-C6H4CH(CH3)(CH2)3, C6H5CH(OCH3)(CH2)2- ' , C6H5CO(CH2)3-, C6H5CO(CH2)4-, (2, 3, or 4)-(alkyl)-C6CH4CO(CH2) 3-, (2, 3, or 4)-(alkyl-oxy)-C6H4CO(CH2)3-, (2, 3, or 4)-X-C6H4 -alkyl-(2, 3, or 4)-X-C6H4CO(CH2)n-, 2-thienyl-CO-(CH2)3-, , (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)D-, Arl-(CH2)n-, C2 to C20 alkyl, X-(CH2)nCO-, or X- (CH2)n-;
n = 1 to 6;
p is l to 20;
X = is independently F, Cl, Br, I, OCH3, SO3-, NH2, H, -OH, -COOH, -COOR, -SO3H, -CN, -NHSO3H, -NO2, or -SO2NH2;
y = H, F, Cl, Br, I, -S03, -PO4=, -OH, -SH, -SCH3, -CH3SO2-, -NH2, or -CO2-; and Ar1 is , independently, aryl, (2, 3, or 4-X-C6H4-), (2, 3, or 4)-(CH2X)C6H4-, (2, 3, or 4)-(CX3)C6H4-, (2, 3, or 4)-(CHX2)C6H4-, 2-thienyl, or (2, 3, or 4)-X-C6H4CH2-;
or its pharmaceutically acceptable salt, including a quaternary ammonium salt.
2. The method of claim 1, wherein the alkyl group is selected from the group consisting of cyclohexyl, (CH3)2CH-, (CH3)2CHCH2-, CH3CH2CH(CH3)-, (CH3)3C-, (CH3)2CH-, and CH3(CH2)n-.
3. The method of claim 1, wherein the Arl group is selected from the group consisting of C6H5-, (2, 3, or 4)-(OCH3)C6H4- and (2, 3, or 4)-(CH3)C6H4-;
2-X-C6H4-, 3-X-C6H4-, and 4-X-C6H4-.
4. The method of claim 1, wherein (2, 3, or 4)-X-C6H4-alkyl- is selected from the group consisting of (2, 3, or 4)-X-C6H4CH(CH2CH3)CH2-, (2, 3, or 4)-X-C6H4CH(CH3)(CH2)-, (2, 3, or 4)-X-C6H4CH(CH3)(CH2)2-, and (2, 3, or 4)-X-C6H4-CH(CH3)(CH2)3-.
5. The method of claim 1, wherein the quaternary ammonium salt is of the formula -NR+Z-, wherein R is alkyl or benzyl, and Z is a counteranion.
6. The method of claim 5, wherein the counteranion is selected from the group consisting of chloride, bromide, iodide, -O-alkyl, toluenesulfonate, methylsulfonate, sulfonate, sulfate, phosphate, and carboxylate.
7. The method of claim 6, wherein the carboxylate is selected from the group consisting of benzoate, succinate, acetate, propionate, glycolate, maleate, malate, citrate, tartrate, ascorbate, benzoate, cinnamoate, mandeloate, benzyloate, and diphenylacetate.
8. The method of claim 1 wherein the mammal is a human.
9. The method of claim 1, wherein the immune response is attributable disorder selected from the group consisting of autoimmune diseases, diseases of unknown etiology having an immunological component, and allergies.
10. The method of claim 4, wherein the autoimmune disease is selected from a group consisting of rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis, psoriasis, leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis, multiple sclerosis, lichen planus, allergic encephalomyelitis, systemic lupus erythematosis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, dry eye associated with Sj?gren's syndrome, 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 lung fibrosis.
11. The method of claim 1, wherein the mammal is treated for allograft rejection.
12. The method of claim 1, wherein the mammal is treated for a graft versus host disease associated with bone marrow transplant.
13. The method of claim 1, wherein the spiperone derivative is administered in a pharmaceutically acceptable carrier.
14. The method of claim 6 wherein the spiperone derivative in combination with an ophthalmic carrier is topically applied to the eye.
15. The method of claim 1, wherein the spiperone derivative is administered systemically.
16. The method of claim %, wherein the dosage is between 0.1 mg/kg to 500 mg/kg of body weight per day as a single daily dose or divided daily doses.
17. The method of claim l wherein the spiperone derivative is administered in a time release formulation.
18. The method of claim 1 wherein the quaternary salt is prepared by combining spiperone or a spiperone derivative with a compound selected from the group consisting of: methyl chloride, methyl bromide, ethyl bromide, methyl iodide, methyl sulfate, ethyl sulfate, methyl benzene-sulfonate, methyl p-toluenesulfonate, ethyl 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.
19. The method of claim 1, wherein the spiperone derivative has decreased serotonin or dopamine receptor binding activity compared to native spiperone and which retains immunosuppressant activity.
20. The method of claim 1, wherein the spiperone derivative is a cycloamylose complex.
21. The method of claim 1, wherein the spiperone derivative is administered in combination with a compound selected from the group consisting of antivirals, antifungals, antibiotics, antiinflammatories, and other immunosuppressants.
22. Pharmaceutical composition comprising an effective amount of a spiperone derivative of the formula:

wherein R1 = H; alkyl, Y-CH2(CH2)n-or Ar1, R2 = H or C1 to C20 alkyl;
R3 = H; alkyl, CN(CH2)2-; X-(CH2)n-; X-(CH2)nCO-;
NH2C(NH)NHC(NH)(aryl)(CH2)n-; or X-(aryl)-(CH2)n-;
R4 = H, C6H5CH(CH2CH3)CH2-, C6H5CH(CH3)(CH2)2-, C6H5CH2CH(CH3)CH2-, C6H5CH2CH2CH(CH3)-, ?6H5CH(CH3)(CH2)3-, 2, 3, or 4)-(alkyl)-C6H4CH(CH3)(CH2)3-, ?2, 3, or 4)-(alkyloxy)-C6H4CH(CH3)(CH2)3, C6H5CH(OCH3)(CH2)2-, , C6H5CO(CH2)3-, C6H5CO(CH2)4-, (2, 3, or 4)-(alkyl)-C6H4CO(CH2)3-, (2, 3, or 4)-(alkyl-oxy)-C6H4CO(CH2)3-, (2, 3, or 4)-X-C6H4-alkyl-(2, 3, or 4)-X-C6H4CO(CH2)n-, 2-thie? CO-(CH2)3-, , (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-, Ar1-(CH2)N-, C1 to C20 alkyl, X-(CH2)nCO-, or X-(CH2)N-;
n = 1 to 6;
p is 1 to 20;
X = is independently F, Cl, Br, I, OCH3, SO3-, NH2, H, -OH, -COOH, -COOR, -SO3H, -CN, -NHSO3H, -NO2, or -SO2NH2;

Y = H, F, Cl, Br, I, -SO3, -PO4=, -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)C6H4-, (2, 3, or 4)-(CHX2)C6H4-, 2-thienyl, or (2, 3, or 4)-X-C6H4CH2-;
or its pharmaceutically acceptable salt, including a quaternary ammonium salt, in a pharmaceutically acceptable carrier.
23. The pharmaceutical composition of claim 22, wherein the alkyl group is selected from the group consisting of cyclohexyl, (CH3)2CH-, (CH3)2CHCH2-, CH3CH2CH(CH3)-, (CH3)3C-, (CH3)2CH-, and CH3(CH2)N-.
24. The pharmaceutical composition of claim 22, wherein the Arl group is selected from the group consisting of C6H5-, (2, 3, or 4)-(OCH3)C6H4- and (2, 3, or 4)-(CH3)C6H4-; 2-X-C6H4-, 3-X-C6H4-, and 4-X-C6H4-.
25. The pharmaceutical composition of claim 22, wherein (2, 3, or 4)-X-C6H4-alkyl- is selected from the group consisting of (2, 3, or 4)-X-C6H4CH(CH2CH3)CH2-, (2, 3, or 4)-X-C6H4CH(CH3)(CH2)-, (2, 3, or 4)-X-C6H4CH(CH3)(CH2)2-, and (2, 3, or 4)-X-C6H4-CH(CH3)(CH2)3-.
26. The pharmaceutical composition of claim 22, wherein the quaternary ammonium salt is of the formula -NR+Z-, wherein R is alkyl or benzyl, and Z is a counteranion.
27. The pharmaceutical composition of claim 26, wherein the counteranion is selected from the group consisting of chloride, bromide, iodide, -O-alkyl, toluenesulfonate, methylsulfonate, sulfonate, sulfate, phosphate, and carboxylate.
28. The pharmaceutical composition of claim 27, wherein the carboxylate is selected from the group consisting of benzoate, succinate, acetate, glycolate, maleate, malate, citrate, tartrate, ascorbate, benzoate, cinnamoat? mandeloate, benzyloate, and diphenylacetate.
29. The pharmaceutical composition of claim 22 in a topical carrier in an amount by weight of between 0.001 and 100% spiperone derivative.
30. The composition of claim 22, wherein the spiperone derivative comprises spiperone complexed with a cycloamylose.
31. The composition of claim 22, wherein the spiperone derivative is in a time release formulation.
32. The composition of claim 22, wherein the spiperone derivative is administered in combination with a compound selected from the group consisting of antivirals, antifungals, antibiotics, antiinflammatories, and other immunosuppressants.
33. The composition of claim 22, wherein the spiperone derivative is provided as a quaternary salt.
34. The composition of claim 22, wherein the spiperone derivative has decreased serot ?in or dopamine receptor binding a ompared to native spiperone and which has imm suppressant activity.
35. The composition of claim 22, wherein the quaternary salt is prepared by combining spiperone or its derivative with a compound selected from the group consisting of methyl chloride, methyl bromide, ethyl bromide, methyl iodide, methyl sulfate, ethyl sulfate, methyl benzene-sulfonate, methyl p-toluenesulfonate, ethyl 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.
CA002126678A 1991-12-27 1992-12-23 Use of spiperone or spiperone derivatives as immunosuppressant agents Abandoned CA2126678A1 (en)

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
US07/815,283 US5290783A (en) 1990-03-16 1991-12-27 Use of spiperone derivatives as immunosuppressant agents
US07/831,429 US5244902A (en) 1989-08-21 1992-02-05 Topical application of spiperone or derivatives thereof for treatment of pathological conditions associated with immune responses
US07/893,534 1992-06-04
US07/831,429 1992-06-04
US07/893,534 US5574041A (en) 1990-03-16 1992-06-04 Use of spiperone derivatives as immunosuppressant agents
US07/815,283 1992-06-04
US07/893,536 US5703088A (en) 1989-08-21 1992-06-04 Topical application of spiperone or derivatives thereof for treatment of pathological conditions associated with immune responses
US07/893,536 1992-06-04

Publications (1)

Publication Number Publication Date
CA2126678A1 true CA2126678A1 (en) 1993-07-08

Family

ID=27505847

Family Applications (1)

Application Number Title Priority Date Filing Date
CA002126678A Abandoned CA2126678A1 (en) 1991-12-27 1992-12-23 Use of spiperone or spiperone derivatives as immunosuppressant agents

Country Status (5)

Country Link
EP (1) EP0661972A1 (en)
JP (1) JPH08500326A (en)
AU (1) AU3421493A (en)
CA (1) CA2126678A1 (en)
WO (1) WO1993012789A1 (en)

Families Citing this family (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5703088A (en) * 1989-08-21 1997-12-30 Beth Israel Deaconess Medical Center, Inc. Topical application of spiperone or derivatives thereof for treatment of pathological conditions associated with immune responses
US5693645A (en) * 1992-12-23 1997-12-02 Beth Israel Deaconess Medical Center, Inc. Use of spiperone or spiperone derivatives as immunosuppressant agents
US5631017A (en) * 1993-03-26 1997-05-20 Beth Israel Deaconess Medical Center, Inc. Topical application of buspirone for treatment of pathological conditions associated with immune responses
US5484788A (en) * 1993-03-26 1996-01-16 Beth Israel Hospital Association Buspirone as a systemic immunosuppressant
US5637314A (en) * 1995-06-07 1997-06-10 Beth Israel Deaconess Medical Center, Inc. Topical and systemic application of buspirone or derivatives thereof for treating atopic dermatitis
CA2240836A1 (en) * 1995-12-21 1997-07-03 Pierre Van Tran Method for treating dermatitis
WO1999059997A1 (en) * 1998-05-18 1999-11-25 Novo Nordisk A/S Novel 1,3,8-triazaspiro[4.5]decanones with high affinity for opioid receptor subtypes
US6277991B1 (en) 1998-05-18 2001-08-21 Novo Nordisk A/S 1,3,8-triazaspiro[4.5]decanones with high affinity for opioid receptor subtypes
CA2496547A1 (en) * 2002-08-29 2004-03-11 University Of Southampton Use of apoptosis inducing agents in the preparation of a medicament for the treatment of liver diseases
AU2003268512A1 (en) 2002-09-09 2004-03-29 Janssen Pharmaceutica N.V. Hydroxy alkyl substituted 1,3,8-triazaspiro[4.5]decan-4-one derivatives useful for the treatment of ORL-1 receptor mediated disorders
CN101622254B (en) 2006-11-28 2013-05-29 詹森药业有限公司 Salts of 3-(3-amino-2-(r)-hydroxy-propyl)-1-(4-fluoro-phenyl)-8-(8-methyl-naphthalen-1-ylmethyl)-1,3,8-triaza-spiro[4.5]decan-4-one
JP5490677B2 (en) 2007-04-09 2014-05-14 ジヤンセン・フアーマシユーチカ・ナームローゼ・フエンノートシヤツプ 1,3,8-Trisubstituted-1,3,8-triaza-spiro [4.5] decan-4-one derivatives as ORL-1 receptor ligands for the treatment of anxiety and depression
US11202776B2 (en) * 2017-05-12 2021-12-21 Takeda Pharmaceutical Company Limited Treatment of gastroparesis with triazaspiro[4.5]decanone

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3155669A (en) * 1962-06-22 1964-11-03 Res Lab Dr C Janssen N V 2, 4, 8-triaza-spiro (4, 5) dec-2-enes
US4839342A (en) * 1987-09-03 1989-06-13 University Of Georgia Research Foundation, Inc. Method of increasing tear production by topical administration of cyclosporin
CA2065375A1 (en) * 1989-08-21 1991-02-22 Richard J. Sharpe Method and composition for the treatment of cutaneous, ocular, and mucosal hypersensitivity, inflammation, and hyperproliferative conditions using topical preparations of serotonin antagonists
WO1991013622A1 (en) * 1990-03-16 1991-09-19 Beth Israel Hospital Association Use of spiperone as an immunosuppressant and anti-inflammatory agent

Also Published As

Publication number Publication date
WO1993012789A1 (en) 1993-07-08
EP0661972A1 (en) 1995-07-12
JPH08500326A (en) 1996-01-16
AU3421493A (en) 1993-07-28
EP0661972A4 (en) 1994-10-27

Similar Documents

Publication Publication Date Title
US5693645A (en) Use of spiperone or spiperone derivatives as immunosuppressant agents
US5290783A (en) Use of spiperone derivatives as immunosuppressant agents
US5703088A (en) Topical application of spiperone or derivatives thereof for treatment of pathological conditions associated with immune responses
US5631017A (en) Topical application of buspirone for treatment of pathological conditions associated with immune responses
US5244902A (en) Topical application of spiperone or derivatives thereof for treatment of pathological conditions associated with immune responses
CA2126678A1 (en) Use of spiperone or spiperone derivatives as immunosuppressant agents
US5637314A (en) Topical and systemic application of buspirone or derivatives thereof for treating atopic dermatitis
RU2469723C2 (en) Therapeutic agent containing carbostiryl derivative and donepezil for treating alzheimer&#39;s disease
US20010006972A1 (en) Nk-1 receptor antagonists for the treatment of symptoms of irritable bowel syndrome
EP0873753A1 (en) Use of NK-1 receptor antagonists for the manufacture of a medicament in the treatment of symptoms of irritable bowel syndrome
CN110167562A (en) DMPC, DMPG, DMPC/DMPG, LYSOPG and LYSOPC are directed to the protective effect for causing the drug of ion channel disease
US5484788A (en) Buspirone as a systemic immunosuppressant
CN116075302A (en) Combinations of GABAA α5 agonists and SV2A inhibitors and methods of use in the treatment of cognitive impairment
US8980922B2 (en) 5-HT4 receptor agonists for the treatment of dementia
KR20110021946A (en) Combination of a nicotinic receptor partial agonist and of an acetylcholinesterase inhibitor, pharmaceutical composition containing same and use thereof in the treatment of cognitive disorders
US5574041A (en) Use of spiperone derivatives as immunosuppressant agents
TW201338772A (en) Pharmaceutical combinations
CA2159091C (en) Topical and systemic application of buspirone or derivatives thereof for treatment of pathological conditions associated with immune responses
EA000531B1 (en) Active substance for production of pharmaceutical preparation for treatment of traumatic brain injury
EP1740176A2 (en) Pharmaceutical compositions comprising anti-inflammatory quinazolinecarboxamide derivatives
US20070191400A1 (en) Pharmaceutical compositions comprising anti-inflammatory quinazolinecarboxamide derivatives
KR20220043046A (en) Use of sphingosine-1-phosphate receptor agonist
WO2017053594A1 (en) Compositions and methods of using the same for decontamination of skin
MXPA02012599A (en) Medicines for the prevention and treatment of neurodegenerative diseases.
CN114761008B (en) Method for treating behavioral and psychological symptoms in patients suffering from dementia

Legal Events

Date Code Title Description
FZDE Dead