Topical application of buspirone for treatment of pathological conditions associated with immune responses

A method for the treatment of a cutaneous, ocular, or mucosal pathological condition which is associated with an immune response in a human or other mammal, that includes topical application of an effective amount of buspirone or a buspirone derivative or its pharmaceutically acceptable salt, optionally in a pharmaceutically-acceptable diluent or carrier for topical application.

BACKGROUND OF THE INVENTION 
This invention is in the area of the topical treatment of cutaneous, 
ocular, and mucosal hypersensitivity and hyperproliferative conditions 
induced by or associated with an immune response, that includes the 
application of an effective amount of buspirone or a buspirone derivative, 
or a pharmaceutically acceptable salt thereof, optionally in a 
pharmaceutically acceptable carrier. 
The immune system specifically recognizes and selectively eliminates 
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 antibodies and/or the destruction of antigenic cells by T lymphocytes; 
both the antibodies and the T lymphocytes are highly specific for the 
antigen or hapten. 
The immune response can provide great benefit to the host when directed 
against an infectious organism. As an example, an important component of 
current public health practices is the use of vaccines to elicit immune 
responses against infectious organisms that cause severe illness and 
death. However, when directed against agents that are relatively 
innocuous, such as pollen, animal dander, and certain plant resins, the 
cells, antibodies, and mediators which represent the effector components 
of the immune response can cause damage to the host's tissues that is out 
of proportion to any threat to health posed by the antigenic agent that 
first elicited the response. 
For example, cutaneous contact hypersensitivity responses are complex 
expressions of cellular immunity characterized by antigen-dependent 
changes in lymphocyte traffic, the recruitment of circulating leukocytes 
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 contact 
hypersensitivity 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 as scarring of affected tissues, itchiness, 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 asthma are just two examples of 
immune responses that can be associated with significant morbidity. Others 
include atopic dermatitis, eczema, 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, 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, neutrophils, and eosinophils) into the skin importantly 
contributes to the pathogenesis of these diseases. Chronic eczema also 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 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. 
It is now believed that leukocytes and other cells found in the normal and 
abnormal skin, eye, or mucosal membranes secrete a variety of cytokines. 
During immunological responses affecting these sites, cytokines are 
important in recruiting additional leukocytes into these tissues, in 
promoting epithelial hyperproliferation, and in inducing other chronic 
changes such as scarring. For example, eosinophils, a type of granulocyte 
found in many pathological immune responses including atopic dermatitis 
and asthma, can produce the cytokine TGF-.alpha. (Wong D. T. W., Weller P. 
F., Galli, S. J., Elovic A., Rand, T. H., Gallagher, G. T., Chiang, T., 
Chou, M. Y., Matossian, K., McBride, J., Todd, R. Human eosinophils 
express transforming growth factor-alpha. J. Exp. Med. 1990; 172:673-81), 
which promotes epithelial hyperproliferation, and TGF-.beta. (Wong, D. T. 
W., Elovic, A., Matossian, K., Nagura, N., McBride, J., Chou, M. Y., 
Gordon, J. R., Rand, T. H., Galli, S. J., Weller, P. F. Eosinophils from 
patients with blood eosinophilia express transforming growth factor 
.beta.1. Blood 1991; 78:2702-2707), which promotes fibrosis. 
In addition to disorders that clearly represent pathological consequences 
of immune responses, immune responses are thought to contribute to many 
other pathological conditions, including Crohn's disease and ulcerative 
colitis of the gastrointestinal tract, psoriasis, alopecia areata and 
others. While the cause of 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 
immune 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 contribute to the 
tissue pathology associated with these disorders, through the production 
of cytokines as well as by 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 therapeutic agents that are both safe and effective. 
Because of the importance of leukocytes and their products in the 
development of pathology associated with immune responses, many approaches 
to 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 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 (PUVA), cyclosporin A, or azathioprine, but the 
risk-to-benefit ratios for these agents is unfavorable for most of the 
conditions described above. 
As a result, there is a significant and very long-standing need to identify 
new agents with 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 and 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. 
In contrast to the immune response, an inflammatory response is a 
pathologic condition that can occur in response to immunologically 
non-specific injury, either from physical (such as trauma), chemical, or 
biologic agents. An inflammatory response is characterized by increased 
blood flow and 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, 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 to or induce an inflammatory response. However, because of the 
differences in mechanisms, a compound can function as an anti-inflammatory 
agent without having immunosuppressive properties. Phenylbutazone, 
indomethacin, aspirin, ibuprofen, and acetaminophen are examples of 
anti-inflammatory compounds which have no significant immunosuppressive 
activity, as demonstrated by their lack of a significant effect on 
immunologically mediated responses, such as contact hypersensitivity. 
PCT International Publication No. WO 91/02527 discloses a method and 
composition to treat cutaneous, mucosal, or ocular hypersensitivity that 
includes administering an effective amount of reserpine, spiperone, or 
other serotonin antagonist. 
Buspirone 
(8-[4-[4-(2-pyrimidinyl)-1-piperaziny]butyl]-8-azaspiro[4.5][decane-7,9-di 
one) is a neuroleptic agent with known central nervous system (CNS) 
dopamine and serotonin (5-HT) receptor antagonist properties. 
It is an object of the present invention to present a method for the 
topical treatment of cutaneous, mucosal and ocular pathology associated 
with immune responses. 
It is yet another object of the present invention to present a method for 
the topical treatment of cutaneous, mucosal, or ocular hypersensitivity 
and epithelial hyperproliferation. 
It is yet another object of the invention to present a method for the 
topical treatment of cutaneous, mucosal or ocular scarring. 
SUMMARY OF THE INVENTION 
A method for the treatment of a cutaneous, ocular, or mucosal condition in 
a human or other mammal resulting from pathology associated with an immune 
response is provided that includes topical application of an effective 
amount of buspirone or a buspirone derivative or its pharmaceutically 
acceptable salt, in a pharmaceutically-acceptable diluent or carrier for 
topical application. 
Buspirone exhibits a strong immunosuppressive activity when applied 
topically. The parent buspirone is used herein as the model of an active 
topical immunosuppressant. Buspirone derivatives are measured against this 
model, and are considered to be immunosuppressants if they suppress the 
ear swelling associated with an experimental contact hypersensitivity 
response by at least 40% at 2 hours after specific antigen challenge. 
In the preferred method of administration, the active compounds are 
administered topically in a suitable carrier in an amount sufficient to 
effectively immunosuppress the patient at the site of application. Because 
the application is topical, i.e., local, immunosuppression is achieved 
without producing significant systemic effects, most notably, the 
significant neuroleptic effect that is associated with the systemic 
administration of buspirone. 
Buspirone and its active derivatives are administered as general 
immunosuppressive agents. The compounds may be 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, aphthous ulcer, iritis, 
conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma, 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 may also be effective 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.

DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS 
The term alkyl, as used herein, unless otherwise specified, refers to a 
saturated straight, branched, or cyclic hydrocarbon of C.sub.1 to 
C.sub.20, 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. 
The term aryl, as used herein, and unless otherwise specified, refers to 
phenyl or substituted phenyl, wherein the substituent is independently 
halo, alkyl, or oxy(alkyl) (for example, methoxy, ethoxy, etc.), and 
wherein the aryl can have up to three substituents. 
The term heterocycle refers to a cyclic moiety that has O, S, or N in the 
aromatic ring, including but not limited to, pyrryl, furyl, pyridyl, 
thiophene, pyrimidyl, thienyl, isothiazolyl, imidazolyl, tetrazolyl, 
pyrazinyl, pyrimidyl, quinolyl, isoquinolyl, benzothienyl, isobenzofuryl, 
pyrazolyl, indolyl, purinyl, carbozolyl, and isoxazolyl and the like, 
optionally substituted with halo (Cl, Br, I, or F), alkyl, oxyalkyl, aryl 
or oxyaryl. 
The term aralkyl refers to an aryl group with an alkyl substituent. 
The term alkaryl refers to an alkyl group that has an aryl substituent. 
The term alkene, as referred to herein, and unless otherwise specified, 
refers to an alkene group of C.sub.2 to C.sub.10, and specifically 
includes vinyl, and allyl. 
As used herein, the term "buspirone" refers to the compound 
(8-[4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl]-8-azaspiro-[decane-7,9-dione 
). 
The term "buspirone derivative" as used herein refers to a compound that 
exhibits an immunosuppressive effect when provided topically, as measured 
using the assay set out in Example 1, i.e., it suppresses the ear swelling 
associated with an experimental contact hypersensitivity response by at 
least 40% at 24 hours after specific antigen challenge, or as evaluated in 
vivo in humans by the agent's ability to inhibit contact hypersensitivity 
responses to patch test allergens in patients hypersensitive to a given 
allergen, using procedures generally accepted by those of skill in the 
art, and wherein the derivative has the formula: 
##STR1## 
wherein: R.sub.1 =H; halo (chloro, bromo, fluoro, or iodo); alkyl, 
specifically including CH.sub.3 --, cyclohexyl, (CH.sub.3).sub.2 CH--, 
CH.sub.3 (CH.sub.2).sub.3 --, (CH.sub.3)CHCH.sub.2 --, CH.sub.3 CH.sub.2 
CH(CH.sub.3)--, (CH.sub.3).sub.3 C--, and --CH.sub.3 (CH.sub.2).sub.p ; 
Y--CH.sub.2 (CH.sub.2).sub.n --; oxyalkyl; or aryl, specifically including 
C.sub.6 H.sub.5 --, (2, 3, or 4)--(OCH.sub.3)C.sub.6 H.sub.4 -- and (2, 3, 
or 4)--(CH.sub.3)C.sub.6 H.sub.4 --; 2--X--C.sub.6 H.sub.4 --, 
3--X--C.sub.6 H.sub.4 --, or 4--X--C.sub.6 H.sub.4 --; oxyaryl; or 
alkaryl; 
R.sub.2 =H, C.sub.6 H.sub.5 CH(CH.sub.2 CH.sub.3)CH.sub.2 --, C.sub.6 
H.sub.5 CH(CH.sub.3)-(CH.sub.2).sub.2 --, C.sub.6 H.sub.5 CH.sub.2 
CH(CH.sub.3)CH.sub.2 --, C.sub.6 H.sub.5 CH.sub.2 CH.sub.2 CH(CH.sub.3)--, 
C.sub.6 H.sub.5 CH(CH.sub.3)(CH.sub.2).sub.3 --, (2, 3, or 
4)-(alkyl)--C.sub.6 H.sub.4 CH(CH.sub.3)(CH.sub.2).sub.3 --, (2, 3, or 
4)-(alkyloxy)--C.sub.6 H.sub.4 CH(CH.sub.3)(CH.sub.2).sub.3, (2, 3, or 
4)--X--C.sub.6 H.sub.4 -alkyl, specifically including (2, 3, or 
4)--X--C.sub.6 H.sub.4 CH(CH.sub.2 CH.sub.3)CH.sub.2 --, (2, 3, or 
4)--X--C.sub.6 H.sub.4 CH(CH.sub.3)(CH.sub.2)--4--X--C.sub.6 H.sub.4 
CH(CH.sub.3)(CH.sub.2).sub.2 --, and 4--X--C.sub.6 H.sub.4 
--CH(CH.sub.3)(CH.sub.2).sub.3 --; C.sub.6 H.sub.5 CH 
(OCH.sub.3)(CH.sub.2).sub.2 --, 
##STR2## 
C.sub.6 H.sub.5 CO(CH.sub.2).sub.3 --, C.sub.6 H.sub.5 CO(CH.sub.2).sub.4 
--, (2, 3, or 4)-(alkyl)--C.sub.6 H.sub.4 CO(CH.sub.2).sub.3 --, (2, 3, or 
4)-(alkyl-oxy)--C.sub.6 H.sub.4 CO(CH.sub.2).sub.3 --, (2, 3, or 
4)--X--C.sub.6 H.sub.4 CO(CH.sub.2).sub.n --, 
2-thienyl--CO--(CH.sub.2).sub.3 --, -alkyl-piperazinyl-aryl; 
-alkyl-C.sub.3-8 cycloalkyl-aryl; -alkyl-piperazinyl-heterocycle; 
-alkyl-C.sub.3-8 cycloalkyl-heterocycle; -alkyl-C.sub.3-8 
cycloalkyl-Ar.sub.1 ; -alkyl-piperazinyl-Ar.sub.1 ; 
-alkenyl-piperazinyl-aryl; -alkenyl-C.sub.3-8 cycloalkyl-aryl; 
-alkyl-aryl-heterocycle; -alkyl-heterocycle-aryl; -alkenyl-C.sub.3-8 
cycloalkyl-Ar.sub.1 ; -alkenyl-piperazinyl-heterocycle; -alkenyl-C.sub.3-8 
cycloalkyl-heterocycle; -alkenyl-piperazinyl-Ar.sub.1 ; 
##STR3## 
(2, 3, or 4)--X--C.sub.6 H.sub.4 C(CH.sub.3)CH(CH.sub.2).sub.2 --, where 
the conformation about the double bond is cis or trans, (2, 3, or 
4)--X--C.sub.6 H.sub.4 C(CH.sub.3)CHCH.sub.2 --, where the conformation 
about the double bond is cis or trans, (2, 3, or 4)--X--C.sub.6 H.sub.4 
COCH.dbd.CHCH.sub.2 --, Y--CH.sub.2 (CH.sub.2).sub.n --, Ar.sub.1 
--(CH.sub.2).sub.n --, C.sub.1 to C.sub.20 alkyl, X--(CH.sub.2).sub.n 
CO--, or X--(CH.sub.2).sub.n --; 
R.sub.3 =.dbd.O, .dbd.NH, .dbd.S, chloro, bromo, iodo, fluoro, alkyl, or 
aryl; 
n=1 to 6; 
p=1 to 20; 
x=is independently F, Cl, Br, I, OCH.sub.3, SO.sub.3, NH.sub.2, H, --OH, 
--COOH, --COOR, --SO.sub.3 H, --CN, --NHSO.sub.3 H, --NO.sub.2, or 
--SO.sub.2 NH.sub.2 ; 
y=H, F, Cl, Br, I, --SO.sub.3, --PO.sub.4.sup..dbd., --OH, --SH, 
--SCH.sub.3, --CH.sub.3 SO.sub.2, --NH.sub.2, or --CO.sub.2 ; and 
Ar.sub.1 =independently, aryl, (2, 3, or 4--X--C.sub.6 H.sub.4 --), (2, 3, 
or 4)--(CH.sub.2 X)C.sub.6 H.sub.4 --, (2, 3, or 4)--(CX.sub.3)C.sub.6 
H.sub.4 --, (2, 3, or 4)--(CHX.sub.2)C.sub.6 H.sub.4 --, 2-thienyl, or (2, 
3, or 4)--X--C.sub.6 H.sub.4 CH.sub.2 --; 
or its pharmaceutically acceptable salt, including any quaternary salt 
known to those in the art, and specifically including the quaternary 
ammonium salt of the formula --NR.sup.+ Z.sup.-, wherein R is alkyl (and 
in particular methyl or ethyl) or benzyl, and Z is a counteranion, 
including chloride, bromide, iodide, --O-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). 
I. Structure and Synthesis of Buspirone Derivatives 
The parent buspirone is 
8-[4-[4-(2-pyrimidinyl)-1-piperaziny]butyl]-8-azaspiro-[4.5]decane-7,9-dio 
ne, which has the structure illustrated below. 
##STR4## 
As demonstrated in Example 1, the parent buspirone has significant 
immunosuppressive activity when applied topically. The potential utility 
of any one of the above-described buspirone derivatives to act as an 
immunosuppressant can be conveniently determined by synthesizing the 
compound and testing it in the biological assay described in Example 1. 
Those derivatives of buspirone which are particularly useful in the method 
of the invention are those which have decreased or no affinities for 
dopamine and/or serotonin receptors, but which retain immunosuppressive 
properties. 
Methods of synthesis of buspirone or its derivatives are disclosed in, or 
can be easily adapted by one of ordinary skill in organic synthesis from 
procedures disclosed in Wu, et al., J. Med. Chem. 15, 477 (1972), Ger. 
Patent No. 2,057,845, and U.S. Pat. No. 3,717,634. See also J. Clin. 
Psychiat. 43, 1-116 (1982). 
II. Therapeutic Compositions 
Mammals, and specifically humans, suffering from pathological cutaneous, 
ocular, or mucosal immune responses can be treated by topical 
administration to the patient of an effective amount of the buspirone 
derivative or its salt, optionally in combination with a pharmaceutically 
acceptable carrier or diluent. 
The active compound is administered topically in an effective dosage range 
to cause immunosuppression of the target pathological immune response. The 
active compound is included in the pharmaceutically acceptable carrier or 
diluent in an amount sufficient to deliver to a patient a therapeutic 
amount of the buspirone derivative in vivo in the absence of serious toxic 
effects. In general, local immunosuppression can be achieved by topically 
administering lower doses of buspirone derivatives than would be required 
if the agents were administered systemically. 
The range of daily dosage for the herein-identified conditions is from 0.01 
grams to 60 grams of topically administered pharmaceutical composition 
containing 0.01% to 10% by weight buspirone. 
Buspirone or its derivative is administered for a sufficient time period to 
alleviate the undesired symptoms and the clinical signs associated with 
the condition being treated. The concentration of active compound in the 
drug composition will depend on absorption, inactivation, and other 
factors known to those of skill in the art. It is to be noted that dosage 
values will also vary with the severity of the condition to be alleviated. 
It is to be further understood that for any particular subject, specific 
dosage regimens should be adjusted over time 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. 
Buspirone or its derivative can be mixed with other active materials which 
do not impair the desired action, or with materials that supplement the 
desired action, such as antibiotics, antifungals, anti-inflammatories, 
antivirals, or other immunosuppressive agents. 
Solutions or suspensions for 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; 
chelating agents such as ethylenediaminetetraacetic acid; buffers such as 
acetates, citrates or phosphates and agents for the adjustment of tonicity 
such as sodium chloride or dextrose. The pH can be adjusted with acids or 
bases, such as hydrochloric acid or sodium hydroxide. 
Suitable vehicles or carriers for topical application are known, and 
include lotions, suspensions, ointments, creams, gels, tinctures, sprays, 
powders, pastes, slow-release transdermal patches, aerosols for asthma, 
suppositories for application to rectal, vaginal, nasal or oral mucosa, 
mouthwashes, or swish and spit preparations. 
Thickening agents, emollients, and stabilizers can be used to prepare 
topical compositions. Examples of thickening agents include petrolatum, 
beeswax, xanthan gum, or polyethylene glycol, 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 and dermatologic applications. 
Natural or artificial flavorings or sweeteners can be added to enhance the 
taste of topical preparations applied for local effect to mucosal 
surfaces. Inert dyes or colors can be added, particularly in the case of 
preparations designed for application to oral mucosal surfaces. 
Buspirone or its derivative can be applied in a time release formulation 
via patches or by slow release polymers. The active compounds can be 
prepared with carriers that will protect the compound against rapid 
release, such as a controlled release formulation, 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. Many methods for the 
preparation of such formulations are patented or generally known to those 
skilled in the art. The materials can also be obtained commercially from 
Alza Corporation and Nova Pharmaceuticals, Inc. 
Buspirone or its 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 toxicological 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, nickel, cadmium, and the like, 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. 
The buspirone derivatives can be modified in order to enhance their 
usefulness as pharmaceutical compositions. For example, it is well know in 
the art that various modifications of the active molecule, such as 
alteration of charge, can affect water and lipid solubility and thus alter 
the potential for percutaneous absorption. The vehicle, or carrier, can 
also be modified to enhance cutaneous absorption, enhance the reservoir 
effect, and minimize potential irritancy or neuropharmacological effects 
of the composition. See, in general, Arndt, K. A., P. V. Mendenhall, "The 
Pharmacology of Topical Therapy", Dermatology in General Medicine, 1987; 
T. B. Fitzpatrick, A. Z. Eisen, K. Wolff, I. M. Freedberg and K. F. 
Austen, eds., 3d ed., McGraw Hill, Inc., New York, pp. 2532-2540. 
III. Immunosuppressant Activity of Buspirone Derivatives 
Buspirone and buspirone derivatives are capable of suppressing the immune 
response in humans and other mammals on topical application. As such, the 
compounds, or therapeutic compositions thereof, may be useful for the 
treatment of a myriad of immunological disorders. 
The ability of buspirone to influence the tissue swelling associated with 
contact hypersensitivity reactions in mice was evaluated as described in 
detail in Example 1. The parent buspirone compound was used for the 
procedure in Example 1 as a model of an active immunosuppressant. 
Buspirone derivatives can be measured against this model, and are 
considered active if they suppress the swelling response by at least 40% 
24 hours after specific antigen challenge. 
Mice treated topically with buspirone, unlike those treated systemically, 
exhibited no significant drowsiness. 
Buspirone expresses both serotonin and dopamine receptor antagonist 
activity. However, unlike buspirone, it was discovered that the chemically 
unrelated serotonin antagonists, trazadone and mianserin, and the dopamine 
receptor antagonist, haloperidol, were not effective in suppressing 
contact hypersensitivity. On the basis of this, it appears that the 
mechanism of action of buspirone on the immune response is independent of 
its serotonin or dopamine receptor blocking properties. 
EXAMPLE 1 
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, N.Y., respectively. 
Buspirone, mianserin, trazadone, haloperidol and oxazolone were purchased 
from the Sigma Chemical Co. (St. Louis, Mo.). 
Oxazolone-Induced Contact Hypersensitivity Sensitization--Sensitization and 
challenge for contact hypersensitivity were performed as follows. The 
abdomens of the mice were shaved with electric clippers, 50 .mu.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 .mu.l of the same solution were applied to each 
hind footpad. Five to eight days later, the mice were challenged for 
contact hypersensitivity by applying 10 .mu.l of a 0.5% (w:w) solution of 
oxazolone in 4:1 (v:v) acetone:olive oil to both the inner and outer 
surface of the right ear of each mouse (in the case of mice treated 
systemically with buspirone) or to both ears (in the case of mice treated 
topically with buspirone). 
Systemic Buspirone Treatment--One hour of after the application of 
oxazolone for elicitation of contact hypersensitivity, mice were treated 
subcutaneously with buspirone 500 or 50 mg/kg body weight) in 0.1 ml of 
carrier (Cremophor EL, BASF, Parsippany, N.J.), or with 0.1 ml of carrier 
alone. In a separate experiment, mice were treated in a similar fashion 
with 50 mg/kg body weight of trazadone, mianserin, haloperidol, or 
buspirone in 1 ml olive oil or with olive oil alone. 
Topical Buspirone Treatment For these experiments, both ears of each mouse 
were challenged for elicitation of contact hypersensitivity by the 
application of oxazolone (as appropriate) to both surfaces of both ears. 
Two hours before, or twenty-four hours after application of hapten, the 
right ears of some mice were treated with buspirone in vehicle, applied 
epicutaneously to both surfaces. The right ears of control mice were 
similarly treated, but with vehicle alone. In the case of experiments 
designed to evaluate the topical effect of buspirone on the sensitization 
phase, only the right ear is challenged (see FIGS. 9 and 10). 
Evaluation of Ear Swelling Response--immediately before and 24 or 48 hours 
after application of oxazolone, ear thicknesses were determined with an 
engineer's micrometer. The increment (delta) in ear thickness (ear 
swelling) was calculated as the 24- or 48-hour value minus the baseline 
(pre-challenge) value and expressed in units of 10.sup.-4 inches. Mice 
were killed by cervical dislocation after the measurement of 24-hour ear 
thickness was obtained, and the ears were processed for histologic 
examination. 
Quantification of Leukocyte Infiltration--Both ears of each mouse were 
fixed in 4.0% buffered formalin and then processed routinely and embedded 
in paraffin for preparation of 6-7 .mu.m-thick hematoxylin and 
eosin-stained sections. All of the sections were coded and examined with 
an ocular grid at 400.times. under light microscopy by an observer unaware 
of the identity of the individual slides. The number of 
leukocytes/mm.sup.2 of dermis was calculated by counting all of the 
leukocyte cells in an area of at least 0.14 mm.sup.2 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). 
Effect of Systemic Buspirone Versus Other Serotonin or Dopamine Receptor 
Antagonists--In these experiments, systemic buspirone was compared to the 
serotonin receptor antagonists, trazadone or mianserin, and to the 
dopamine receptor antagonist, haloperidol, for their ability to inhibit 
cutaneous contact hypersensitivity. At a dose of 50 mg/kg, only buspirone 
significantly reduced cutaneous contact hypersensitivity (FIG. 1, 2). 
EXAMPLE 2 
Comparison of Immunosuppressant Versus Anti-Inflammatory Activity. 
Mice were sensitized to oxazolone as described in Example 1. 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. 90:311 (1988). 
Three days later, mice were challenged for contact hypersensitivity as in 
Example 1. When the hypersensitivity response was assessed 24 hours later, 
indomethacin was shown to have no significant effect on the response. A 
classic anti-inflammatory agent, indomethacin, does not appear to suppress 
the edema associated with the immunologically specific oxazolone induced 
contact hypersensitivity response and compared to buspirone, only weakly 
suppresses the leukocyte infiltration associated with the response. 
EXAMPLE 3 
Evaluation of Serotonin Receptor Binding Activity or Dopamine Receptor 
Binding Activity of Buspirone Derivatives. 
Buspirone 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 commercially can be 
used as the ligand. The buspirone derivative to be tested is then 
incubated with the radiolabeled quipazine ligand combination. Displacement 
of radiolabeled ligand is positive evidence that the buspirone 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 .sup.3 
H-quipazine and the example follows: 
Binding studies using .sup.3 H-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.degree. C. and centrifuged at 49,000.times. g for 10 
min. The pellet is resuspended in fresh buffer and incubated at 37.degree. 
C. for 10 min. After the final centrifugation, the pellet is resuspended 
in 80 volumes of Krebs-HEPES buffer (25 mM HEPES, 118 mM NaCl, 5 mM KCl, 
2.5 mM CaCl.sub.2, and 1.2 mM MgCl.sub.2 pH adjusted to 7.4). Tissue (10 
mg of original wet weight) is added to assay tubes containing 0.8 nM 
[.sub.3 H]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 5 ml 
of 50 mM Tris-HCl buffer pH 7.7. Radioactivity is quantified by liquid 
scintillation counting. 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. 
Buspirone derivatives which have binding affinities for dopamine and/or 
serotonin receptors of one/tenth or less than the parent buspirone are 
considered to be potentially useful as systemic immunosuppressants if they 
are at least 50% as active as the parent buspirone on a weight basis in 
suppressing immunologically specific responses such as contact 
hypersensitivity. 
Modifications and variations of the present invention relating to methods 
for the treatment of pathology associated with immune responses that 
includes topical administration of an effective amount of buspirone or a 
buspirone derivative 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.