Methods for inducing site-specific immunosuppression and compositions of site specific immunosuppressants

The present invention provides methods and formulations for site-specific immune suppression of immune/inflammatory responses with localized or topical application of immunosuppressants including cyclosporines, rapamycins (RPM), or combinations of immunosuppressants and anti-inflammatory compounds. Methods for the use of said formulations to effect site-specific immune suppression of local inflammatory/immune responses in mammalian tissue and for treatment of autoimmune, T-cell mediated immune disease, inflammatory conditions, inhibition of contact hypersensitivity, and for producing prolonged skin allograft survival, and wound healing are presented. In addition, methods for use of said formulations--in tandem with systemic applications of immunosuppressant such as cyclosporine or without same--are presented. The present invention also relates to alternative formulations and delivery systems for the efficacious treatment of the aforementioned conditions.

Cyclosporine (CsA), a selective immunosuppressant and a potent 
anti-inflammatory agent, has demonstrated great clinical success in 
inhibiting T-cell mediated immune processes such as allograft rejection, 
graft-versus-host disease, and autoimmune disease when administered 
systemically. (See, e.g., A. D. Hess al., Transpl. Proc. 20: 29 (1988).) 
As to the latter, systemic CsA has been proven efficacious for treating 
psoriasis, an autoimmune disorder of the skin. (See, e.g., C. N. Ellis, et 
al., JAMA 256: 3110 (1986).). However, the induction of immunosuppression 
at the tissue site and focal responding immunocytes could result in 
surprisingly greater efficacy, and could have significant immunologic and 
clinical ramifications. 
As an example of the aforementioned ramifications, within the specialty of 
dermatology, it would be desirable to treat putative autoimmune conditions 
and related diseases of the skin, including, for example, eczema, contact 
hypersensitivity, alopecia areata and psoriasis. Few if any models for 
testing the disease mechanism and the efficacy of various treatment 
modalities have been available in this field, however. Moreover, due to 
the variability of expression of most skin conditions, and the inherent 
differences between epidermal tissues in various locations on the body, a 
single treatment methodology or pharmaceutical composition is rarely 
effective for all disease conditions presented. 
A basic understanding of the immune response involved will facilitate the 
understanding and appreciation of the present invention. T-cell mediated 
immune events play an important role in eliciting allograft rejection and 
other inflammatory reactions. The immunological cascade that follows 
alloengraftment includes: (1) recognition of antigen; (2) lymphocyte 
activation; (3) development of specific cellular and molecular lines of 
communication between responding immunocytes via lymphokine release and 
induced expression of major histocompatibility complex ("MHC") antigens; 
and (4) mononuclear inflammatory cell infiltration into the target tissue 
which leads to eventual graft destruction (rejection). Systemic 
administration of CsA, a novel fungal metabolite, is well known to block 
this inflammatory cascade and to facilitate permanent allograft acceptance 
(actively-acquired immunological tolerance) in various experimental animal 
models, probably by inhibitory effects upon T-helper cells with sparing of 
T-suppressor cell expression. (See, e.g., A. D. Hess, et al., Transpl. 
Proc. 29 (1988).) Cyclosporines and other similar immunosuppressants such 
as rapamycins, FK-506 derivatives and immunophilin binding agent have 
novel immunosuppressive properties compared to conventional agents: they 
are selective in their mechanism of action, demonstrate superior graft 
survival times, and are potent anti-inflammatory compounds. Cyclosporines 
are well-recognized for their powerful ability to permanently alter immune 
responsiveness, in comparison with conventional agents, so that some 
degree of selective immunologic tolerance (graft acceptance) can be 
achieved in various models. Therefore, it would be extremely advantageous 
and desirable to develop topical formulations of cyclosporines, rapamycins 
and other immunosuppressants for localized tissue site-specific action. 
Conventionally, immunosuppressants have been administered at a systemic 
level in order to inhibit both cell- and humoral-mediated immune 
responses. However, the induction of localized site-specific 
immunosuppression could inhibit the mechanisms which lead to graft 
rejection and similar inflammatory immune processes operative in 
autoimmune and putative autoimmune disorders. Yet, a tissue site-specific 
immunosuppressive mechanism has not been conclusively demonstrated by 
local application of the cyclosporines. 
More recently, the fungal metabolites known as cyclosporines, and 
particularly Cyclosporine A (CsA), have been established as the principal 
immunosuppressants in solid organ transplantations. The systemic use of 
cyclosporine prolongs the survival of experimental and clinical 
allografts, but continuing immunosuppressive therapy is generally 
necessary. 
Yet, the long-term side effects of systemic administration of cyclosporines 
and other immunosuppressants are of major concern. The related 
complications of nephrotoxicity and hepatotoxicity (i.e., kidney and liver 
damage), as well as an increase in infections, are a significant problem 
and may thus render treatment with cyclosporines inappropriate for certain 
patients, such as those who have been severely burned, or for those with 
skin conditions that are not life-threatening, such as psoriasis. One 
method for achieving indefinite survival of the graft or prolonged 
anti-inflammatory effects with CsA and/or other immunosuppressants and for 
reducing potentially toxic systemic side effects involves the localization 
of CsA and/or other immunosuppressants in the target tissue. 
For the purposes of clarity and easier comprehension, the terms "CsA", 
"Cyclosporine A" and "cyclosporine" may be considered interchangeable with 
the term "cyclosporin(s)" throughout this disclosure. While CsA is the 
cyclosporine typically used in most pharmaceutical preparations, the scope 
of this invention is not limited to this one type of cyclosporine. 
Likewise, the terms "rapamycin", "RAP", "RPM", "rapamycin derivatives", 
and "rapamycin prodrugs" may be considered interchangeable with the term 
"rapamycin(s)" throughout this disclosure. Similarly, the terms "steroid", 
"anti-inflammatory hormone", "corticosteroid anti-inflammatory", 
"corticosteroid", "glucocorticoid anti-inflammatory", "glucocorticoid", 
"steroid anti-inflammatory" and "steroid immunosuppressant" may be 
considered interchangeable throughout this disclosure. 
Local inhibition of the rejection response with CsA has demonstrated mixed 
results. Perfusion of kidney allografts with CsA prior to transplantation 
did produce enhancement of tissue survival; however, prior, minimal 
systemic azathioprine immunosuppression was required. See, e.g., L. H. 
Toledo-Pereyra, et al., Transplantation 33:330 (1982). Likewise, infusion 
of low-dose CsA into the ligated thoracic duct provided only a mild 
enhancement of rat kidney allograft survival. Delayed type 
hypersensitivity has been effectively inhibited in animals and man with 
topically-applied CsA (see, e.g., R. D. Aldridge, et al., Clin. Exp. 
Immunol. 59: 23, 1985), as has cornea allograft rejection. The topical 
application of CsA has also been shown to be effective in treating 
alopecia areata and contact hypersensitivity in humans, yet it appears to 
have no effect on psoriasis. Studies using topically-applied CsA 
demonstrated prolonged survival of rat skin allografts; see, e.g., C. S. 
Lai, et al., Transplantation 44: 83, 1987, X. F. Zhao, et al., Transplant. 
Proc. 20: 670 (1988). However, one such study concluded that most of the 
enhancement observed with local CsA treatment was due to the animals' 
ingestion of CsA from the treated area. See Zhao, supra. When means were 
taken to prevent the animals from ingesting CsA from the grafts, the 
investigators found that CsA blood levels were suboptimal (below 100 
ng/ml) and negligible enhancement of skin allograft survival was seen. It 
has also been postulated that autoimmune disorders of the skin could 
benefit from transdermal (i.e., localized) treatment with CsA. 
Thus, there is a need for topical and local formulations of 
immunosuppressants, particularly those that react with immunophilin 
cytosolic binding proteins, which include but are not limited to 
cyclosporines, rapamycins, FK 506 derivatives and prodrugs, and 
combinational immunosuppressants. There is also a need for a method for 
utilizing same, in the prevention of localized tissue site-specific 
inflammatory immune reactions. An example includes prevention of skin 
allograft rejection and contact hypersensitivity reactions at a local 
level, but these would serve as models for other inflammatory disorders 
such as autoimmune disease of the skin (i.e., psoriasis, lupus, contact 
hypersensitivity, alopecia areata, dermatitis, dermatoses), localized 
tissue auto- or allo- inflammatory/immune responses, and tissue or organ 
allografts. In particular, a methodology that locally provides allograft 
acceptance and attenuates T-cell mediated events is highly desirable. The 
present invention is directed to such formulations and methods of use. 
Immunosuppressants represent a revolutionary new class of potent 
anti-inflammatory agents possessing selective actions and reduced 
side-effects. Their mechanism of action is not completely known. However, 
such immunosuppressants derived from microorganisms including the 
cyclosporines, and macrolides such as FK506, Rapamycin and derivatives 
possess common properties. They are lipophilic antibiotics that inhibit 
the transcription of T cell activation genes and/or signal transduction 
pathways involved in T cell activation. A class of cytosolic binding 
proteins for these agents have been identified (immunophilins), which are 
peptidyl-prolyl-cis- trans-isomerases and have been implicated in 
signaling pathways for T cell activation. 
The status of the immune system must be considered as either activated 
(primed) or inactivated (resting). Some immunosuppressive compounds may 
preferentially affect immune mechanisms in the resting state to inhibit 
progressive activation. However, other agents may be at a disadvantage in 
inhibiting a preactivated immune response. Cyclosporine has been shown to 
provide potent and selective immunosuppression in the preactivation phase 
of in vitro and in vivo models. A prominent mechanism of action for 
cyclosporine and other similar immunosuppressants is inhibition at the 
level of the CD4+ MHC class II responsive helper T cell. In particular, 
cyclosporine inhibits the release of cytokines such as IL-2 and allows 
expression of antigen specific suppressor circuits. 
One of the most fascinating and important aspects of the cyclosporines and 
other immunosuppressants is that they are well-known to induce some degree 
of permanent attenuation of immune responsiveness. This is known as 
induction of immunological tolerance. Research in our laboratories and 
others have supported this conclusion. This fact alone makes cyclosporine 
an exciting and novel candidate drug for auto-immune and inflammatory 
diseases along with other similar immunosuppressants. 
Yet, systemic immunosuppressant administration is of great concern due to 
dangerous unwanted side-effects. Thus, it would be extremely advantageous 
to effect site-specific immunosuppression with the cyclosporines or other 
similar immunosuppressants by targeting the drug to specific tissue sites. 
Targeting the drug to the site where it is most needed would help overcome 
this concern and increase efficacy. However, the cyclosporines are 
generally known to be primarily effective only during induction of immune 
responsiveness, not following activation of immunity. Therefore, due to 
the known mechanisms of action for these novel immunosuppressants, it 
would not necessarily be hypothesized that such agents could be effective 
in autoimmune disease states where activation is already occurring. Yet, 
most surprisingly, and in contradistinction to these known mechanisms, we 
have demonstrated that dramatic site-specific efficacy can indeed be 
achieved with cyclosporine using site-specific application. 
Specifically, new research has proven that inflammatory reactions in the 
skin can be inhibited at the tissue site using topical formulations of 
immunosuppressants. Details concerning topical drug formulations and the 
critical methodology for their successful use are provided herein. The 
basic technology of site-specific immunosuppression involves: the 
physicochemical properties of immunosuppressants related to drug delivery 
and targeting to specific tissue sites; and immune principles discovered 
that are necessary for inhibiting activated immune responses by 
cyclosporine, rapamycin, and other immunosuppressants during a disease 
state. 
SUMMARY OF THE INVENTION 
The present invention exploits observations that: 1) cyclosporine and 
rapamycin inhibit primary inflammatory/immune responses by local 
application using in vitro cellular site-specific models; 2) cyclosporine 
and rapamycin inhibit activated inflammatory/immune responses by local 
application using in vitro cellular site-specific models; 3) rapamycin is 
surprisingly efficacious with local application during both late and early 
inflammatory immune phases using in vitro cellular site-specific models; 
4) cyclosporine is more efficacious locally during the early inflammatory 
immune phase compared to the late phase using in vitro cellular 
site-specific models; 5) consistent with these in vitro findings, either 
cyclosporine or rapamycin inhibit local inflammatory/immune responses by 
topical application to skin tissue using in vivo models of site-specific 
immune suppression; 6) this includes site-specific immune suppression 
effected by topical use of cyclosporine and rapamycin combinations in 
contact hypersensitivity reactions of skin tissue; 7) rapamycin is 
particularly efficacious during the late local inflammatory-immune phase 
in this latter example; 8) cyclosporine is particularly efficacious during 
the early local inflammatory immune phase in this latter example; and 9) 
in agreement with these results, skin allograft survival may be prolonged 
via topical use of cyclosporines, alone and in combination with other 
anti-inflammatory agents, and more particularly, with Cyclosporine A alone 
or combined with steroidal anti-inflammatory agents such as hydrocortisone 
to produce synergistic results. 
The present invention is based on the concept that targeting CsA, RPM, 
other immunosuppressants, or combinations of immunosuppressants and 
anti-inflammatory compounds to a specific tissue is a desirable means for 
increasing efficacy and reducing systemic toxic concerns associated with 
these immunosuppressants. This localized effect of immunosuppressants also 
indicates potential usefulness in organ transplants, via perfusion and/or 
topical application. Further, immunosuppressants may be effective in the 
clinical treatment of autoimmune skin disorders and other localized 
inflammatory reactions. In general, then, this treatment may be 
appropriate whenever there is a T-cell-mediated or mononuclear cellular 
inflammatory reaction incited by a fixed-tissue-based antigen and/or 
unknown mechanisms. In addition, local application with cyclosporines, 
RPM, immunosuppressants, or combinations of immunosuppressants and 
additional anti-inflammatory agents may prove efficacious for the 
treatment of rheumatoid arthritis, osteoarthritis, temporal-mandibular 
joint disease (TMJ), asthma, multiple sclerosis, colitis, ophthalmic 
inflammatory conditions, uveitis, meningitis, inflammatory bowel disease, 
myositis, inflammation of oral and esophageal tissues, inflammatory lung 
disease, inflammation associated with myocardial infarction and cerebral 
vascular disease or accidents, trauma induced inflammation, and other 
inflammatory/immune disorders. 
A critical mechanism for the induction of site-specific immune suppression 
by immunosuppressants appears to be the establishment of a systemic 
maintenance phase of immune nonresponsiveness. To induce this maintenance 
state, an initial limited systemic dose of CsA or another 
immunosuppressant appears necessary. Analogously, it is well-recognized 
that two distinct states of immunosuppression, the induction and 
maintenance phases, are important for the development of specific immune 
non-responsiveness. (See, e.g., E. Towpik, et al., Transplantation 40: 714 
[1985]). It is not unlikely that immunosuppressant, dosing requirements 
for efficacious site-specific suppression of autoimmune inflammatory skin 
disorders will underscore this observation. Either continuous or limited 
low-dose immunosuppressant administered systemically at various times in 
conjunction with topical application may also prove efficacious. 
Additionally, transdermal delivery of immunosuppressants thereby providing 
systemic and local effects may also prove efficacious. 
In accordance with one aspect of the present invention, there is provided a 
method for utilizing local CsA, in a topical formulation in conjunction 
with a short-term, limited systemic CsA schedule or a longer-term, 
low-dose systemic CsA schedule for effective abrogation of skin allograft 
rejection, T-cell mediated immune processes, and inflammatory reactions. 
This method should also prove effective in the clinical treatment of 
autoimmune skin disorders including psoriasis and other localized 
inflammatory reactions or cyclosporine-responsive conditions. One 
preferred embodiment suggests a-systemically applied formulation wherein 
about 1 mg/kg/day to 25 mg/kg/day of cyclosporine is applied per single 
dosage. 
In accordance with another aspect of the present invention, there is 
provided a method for utilizing local CsA in combination with additional 
anti-inflammatory agents, such as steroids or hydrocortisone, in a topical 
formulation for synergistic abrogation of skin allograft rejection, T-cell 
mediated immune processes, and inflammatory reactions. This method should 
also prove effective in the clinical treatment of autoimmune skin 
disorders including psoriasis and other localized inflammatory reactions 
or immunosuppressant-responsive conditions. 
In accordance with another aspect of the present invention, there is 
provided a method for utilizing local rapamycin in a topical formulation 
for efficacious abrogation of skin hypersensitivity reactions, T-cell 
mediated immune processes, and inflammatory reactions. This method should 
also prove effective in the clinical treatment of autoimmune skin 
disorders including psoriasis and other localized inflammatory reactions 
or immunosuppressant-responsive conditions. 
In accordance with another aspect of the present invention, there is 
provided a method for utilizing local CsA in combination with a 
immunosuppressant agent, rapamycin, in a topical formulation for 
efficacious abrogation of skin hypersensitivity reactions, T-cell mediated 
immune processes, and inflammatory reactions. Novel combinations of 
immunosuppressive agents such as rapamycin and cyclosporine enable 
differential actions on immunoactivation pathways for potential synergism. 
This method should also prove effective in the clinical treatment of 
autoimmune skin disorders including psoriasis and other localized 
inflammatory reactions or immunosuppressant-responsive conditions. 
In one embodiment, CsA is suspended in a topical cream formulation of a 
particular composition. In another embodiment, CsA is a component of a 
mineral oil-based topical formulation of a particular composition. In 
accordance with yet another embodiment of the invention, a topical 
formulation of cyclosporine is provided wherein CsA is embodied in a 
jojoba oil-based topical formulation of a particular composition. In 
accordance with other embodiments, the formulation is embodied in a paste, 
a gel, a liquid or a spray. Additionally, other embodiments include 
topical formulation of CsA in conjunction with different 
immunosuppressants and anti-inflammatory agents. Additional embodiments 
include formulations containing a preservative, as well. 
For example, one preferred type of formulation according to the present 
invention may generally comprise cyclosporine, a pharmaceutical carrier, a 
co-solvent, a penetration enhancer, and an emulsifier. In a further 
embodiment, said components may be present in these approximate 
quantities: 5-80% pharmaceutical carrier; 5-50% co-solvent; 1-5% 
penetration enhancer; 0.1-20% emulsifier; and 0.2-25% cyclosporine (or 
cyclosporine applied to the tissue in such an amount that from about 0.5 
mg/cm.sup.2 to 5 mg/cm.sup.2 of cyclosporine is applied per single dose). 
Another preferred type of formulation according to the present invention 
may generally comprise, in approximate amounts by weight, 5-60% anhydrous 
lanolin; 5-60% mineral oil; 5-60% olive oil; 5-30% ethyl alcohol; 5-50% 
deionized water; 5-15% glycerol; 0.2-20% polysorbate 80; 1-5% 
polyvinylpyrrolidone; 0.2-25% cyclosporine A powder; and 0.1-10% sodium 
dodecyl sulfate. 
Still another preferred type of formulation according to the present 
invention may generally comprise, in approximate amounts by weight, 5-60% 
anhydrous lanolin; 5-80% jojoba oil; 5-80% olive oil, 0.2-20% polysorbate 
80; and 0.2-25% cyclosporine A powder. 
An additional preferred type of formulation according to the present 
invention may generally comprise, in approximate amounts by weight, 5-60% 
anhydrous lanolin; 5-80% white petrolatum; 5-80% olive oil; 0.2-20% 
polysorbate 80; and 0.2-25% cyclosporine A powder. 
Another preferred type of formulation according to the present invention 
may generally comprise, in approximate amounts by weight, 5-60% anhydrous 
lanolin; 5-80% white petrolatum; 5-80% olive oil; 0.2-20% polysorbate 80; 
and 0.2-25% cyclosporine A powder. 
Another preferred type of formulation according to the present invention 
may generally comprise, in approximate amounts by weight, 60-90% ethyl 
alcohol; 3-30% glycerol; 0.2-20% polysorbate 80, and 0.2-25% cyclosporine 
A powder. 
According to the present invention, yet another example of a preferred 
formulation generally comprises, in approximate amounts by weight, 0-50% 
ethyl alcohol (v/v); 5-30% glycerol (v/v); 10-90% propylene glycol (v/v); 
and 0.2-25% cyclosporine A powder (w/v). 
Another preferred type of formulation according to the present invention 
may generally comprise, in approximate amounts by weight, 0.2-20% 
polysorbate 80 (v/v); 2-30% ethyl alcohol (v/v); 5-50% deionized water 
(v/v); 5-40% glycerol (v/v); 10-80% propylene glycol (v/v); and 0.2-25% 
cyclosporine A powder (g/100 ml; w/v). 
Another preferred type of formulation according to the present invention 
may generally comprise, in approximate amounts by weight, 0-20% ethanol 
(v/v); 0.2-25% cyclosporine (w/v); 19-80% white petrolatum (v/v); 0-10% 
heavy mineral oil (v/v); and 0.05-5% steroid powder (w/v). A further 
embodiment may utilize hydrocortisone as the steroid powder of choice. 
Yet another preferred type of formulation according to the present 
invention may generally comprise cyclosporine and a pharmaceutically 
acceptable pharmaceutical carrier. Such a formulation may further comprise 
an esterification product of natural triglycerides and polyethylene 
glycol; a vegetable oil; and ethanol. 
Another preferred type of formulation according to the present invention 
may generally comprise a formulation wherein the weight ratio of ester to 
cyclosporine is about 10:0.2 to 10 parts by weight; vegetable oil is about 
35 to 60% of the total composition by weight; and ethanol is about 1 to 
20% of the total composition by weight. Further, such a formulation may 
generally include cyclosporine, wherein the cyclosporine is cyclosporine A 
powder in a concentration by weight of about 0.5% to about 25%. 
In accordance with another aspect of the present invention, a dual skin 
graft model is provided, which may be used, for example, to test treatment 
protocols, such as the tandem treatment method suggested herein, or the 
topical administration of various cyclosporine-containing formulations. 
Further, the present invention proposes that the use of pharmaceutically 
acceptable co-solvents and potential penetration promoters in 
cyclosporine-containing topical treatment formulations that may result in 
decreased or lost efficacy locally, but increased efficacy systemically. 
Therefore, a gradient effect may be created by such formulations in the 
locally-treated tissues which extends into the systemic circulation. 
However, by lowering cyclosporine doses with such formulations, the 
potentially desired local result can be effected. In contradistinction, 
topical cyclosporine formulations without said co-solvents and obvious 
penetration promoters generally appear to facilitate deposition of the 
active agent locally in the treated tissues. These latter formulations are 
more effective at producing only localized effects without systemic 
involvement at equivalent cyclosporine concentrations. 
In addition, it is suggested that various combinations of cyclosporines, 
steroids and other anti-inflammatory agents (non-steroidal agents, for 
example) be used in the local treatment of autoimmune and other 
inflammatory conditions to provide combined, additive, and/or synergistic 
efficacy. 
In another embodiment of the present invention, alternative delivery 
systems, such as microencapsulation of cyclosporine-containing 
formulations within lipid membranous vesicles such as liposomes, or 
microemulsions are suggested. 
Other embodiments of the present invention include the effective 
administration of CsA for systemic purposes via transdermal application. 
It is thought that this novel route of administration of CsA may provide 
new mechanisms of systemic action of CsA due to different metabolism when 
cyclosporine passes through the epidermis/dermis. These results also 
support the use of topical CsA formulations as an effective means for 
systemic delivery in patients needing immunosuppression but who may 
present compromised gastrointestinal absorption. 
In addition, it is suggested, in another embodiment, that CsA may be 
administered locally to various tissues other than the skin; e.g., to the 
oral mucosa, the esophagus, the nasal septum, the bronchical tubes, and 
lung tissue, to name a few. 
Moreover, CsA has been shown to have mild antifungal properties and topical 
application may be effective for fungal infections. Such application is 
suggested in another embodiment of the present invention. 
Finally, the present invention proposes a method for utilizing any one of 
several topical CsA, RPM, combined CSA-RPM, combined immunosuppressant, or 
combined immunosuppressant/anti-inflammatory formulations in conjunction 
with systemically-applied CsA, or independently of same. Examples of 
substances which may be used as co-solvents in the illustrated embodiments 
include the following: ethanol; oleyl alcohol; alkylene polyols; glycerol; 
polyethylene glycol; oleic acids, vegetable oil PEG-6 complexes; caprylic 
triglyceride; capric triglyceride; glyceryl caprylate; glyceryl caprate; 
PEG-8 caprylate; PEG-8 caprate; ethoxydiglycol; and any mixture thereof. 
Examples of substances which may be used as penetration enhancers in the 
illustrated formulations include the following: ethanol; oleyl alcohol; 
alkylene polyols; oleic acids; urea; pyrrolidones; surfactants such as 
sodium lauryl sulfate; vegetable oil PEG-6 complexes such as the 
commercially available Labrafils (Gattefosse, Elmsford, N.Y.); 
caprylic/capric triglyceride (i.e., Labrafac Hydro, Gattefosse); glyceryl 
caprylate/caprate and PEG-8 caprylate/caprate (Labrasol, Gattefosse); and 
ethoxydiglycol (i.e., Transcutol, Gattefosse) caprylic triglyceride, 
capric triglyceride; glyceryl caprylate; glyceryl caprate; PET-8 
caprylate; PEG-8 caprate; and any mixture thereof. 
One advantage of the present invention over the prior art includes the fact 
that topical application of cyclosporine is effective in abrogating skin 
allograft rejection, inflammatory reactions and autoimmune skin disorders, 
without interfering with other cellular processes. As noted previously, 
other topically-applied formulations, such as those containing steroids, 
exclusively are less efficacious immunosuppressants, are less selective in 
their actions, and are less effective at inducing permanent immunologic 
tolerance than are cyclosporines or similar immunosuppressants. Further, 
in the case of steroid creams and ointments used exclusively, a 
detrimental effect on wound healing and non-specific immunity against 
infection may result from their use. 
A further advantage of the present invention is the fact that selectively 
delivering cyclosporine or immunosuppressant to a specific tissue targets 
the compound to responsive inflammatory cells and is a desirable means of 
increasing efficacy and reducing systemic toxic concerns associated with 
immunosuppressants, in that the localized effect of cyclosporine indicates 
that it is potentially useful in organ transplants via topical application 
and/or via perfusion. Topical application of cyclosporine promotes 
allograft survival by delivering the compound to the target tissue, which 
facilitates the site-specific activity and efficacy of this 
immunosuppressant, while reducing potentially toxic systemic levels of 
cyclosporine. 
Another advantage of the present invention is the fact that the dual skin 
allograft model provides an excellent research and clinical study 
protocol. For example, use of two allografts, one receiving treatment and 
the other left untreated, allows in vivo assessment of the systemic T-cell 
mediated response against the particular allograft in question. Since the 
treated allograft will potentially elicit systemic alloactivation, 
assessment of the test substance's ability to locally suppress these 
systemic alloaggressive cells will be possible. In addition, local effects 
of a test substance may be studied via the proposed dual skin allograft 
model. 
Further advantages include the efficacy of the invention in treating a 
disease such as alopecia, where relatively normal skin is receiving 
treatment. In such instances, the required formulation is likely to be 
different from that which would effectively treat a more severe skin 
disorder such as psoriasis complicated by open lesions. In addition, dose 
and timing requirements will require study of the patient by the 
practitioner, and may necessitate variations for both systemic and topical 
phases of treatment with immunosuppressants. 
Likewise, some conditions may require topical immunosuppressant application 
alone, without prior systemic CsA treatment. Moreover, different 
formulations may easily be devised according to the protocols and methods 
set forth herein, to produce creams or ointments which may prove 
efficacious and advantageous.

Experiments confirm that there are two important aspects which must be 
considered when investigating local immunosuppressive therapy: immune 
function and pharmacology. Data discussed below deals exclusively with 
either 1) local interference of immune and inflammatory activation 
mechanisms (immune function); 2) pharmacology of local immunosuppressant 
delivery to specific tissue sites by topical application; or 3) combined 
immune functional and pharmacologic considerations. 
Immunology of Site-specific Immune Suppression 
One can assume in autoimmune and inflammatory diseases there exists a state 
of immune activation. Under these conditions cyclosporine may 
theoretically be limited to therapy in disease states consistent with 
known mechanisms of cyclosporine immunosuppression where immune activation 
is being initiated. However, both in vitro and in vivo data within our lab 
demonstrates that CsA does indeed have potent immunosuppressive effects on 
preactivated cells, as concentrations are increased to relatively high 
values locally (see FIG. 1 A, B and C). 
In FIG. 2, topical CsA provided moderate graft prolongation and disparity 
in a vehicle system that provided less-than-optimal transepidermal 
delivery and graft prolongation and was designed to test for combinational 
immunosuppressant synergism (Bar 1). Mean survival times increased 
slightly with combined topical CsA/HC in comparison to CsA alone, but, did 
not provide a significant synergism (Bar 2). However, topical CsA during 
immune response induction or the antigen-dependent phase with subsequent 
suppression of antigen-independent inflammation by topical CsA/HC provided 
dramatic synergism with optimal efficacy and disparity (Bar 3). In FIG. 3, 
systemic profiles of serum CsA yielded a predictable trend in placebo 
treated skin allograft survival. Ten day subcutaneous treatment reached a 
peak of 1,700 ng/ml at day 11. Subtherapeutic levels were reached and 
maintained by day 25. Placebo grafts rejected shortly thereafter. 
High CsA levels in the systemic circulation would normally be expected to 
produce organ toxicity. However, high levels of CSA can be localized at 
the tissue site undergoing immune activation without adverse effects (see 
test systems below and FIG. 4). 
In FIG. 4, the skin allografts demonstrated very high tissue levels in the 
treated graft (37.5.+-.25.3 ug/g), while the control grafts demonstrated 
relatively low tissue levels (1.6.+-.0.6 ug/g) at 36 days 
post-transplantation. These results were obtained with a formulation that 
produced site-specific efficacy in vivo, using a vehicle system comprising 
lanoline: mineral oil: olive oil: polysorbate 80 (7:7:7:4) and CsA (2.5% 
w/v). At this time, placebo grafts were undergoing vigorous rejection. The 
treated graft contained 2,629% greater CsA than the vehicle treated graft. 
Animal experiments show that local CsA is efficacious and non-toxic. 
Systemic application is necessary to modulate the immune response in 
accord with the known mechanisms of actions of cyclosporine (see FIG. 5). 
In addition, as wound healing is expedited with systemic administration, a 
nidus of inflammation is therefore avoided. 
The results in tissue culture systems and in vivo demonstrate that prior 
systemic cyclosporine treatment sensitizes both basal and activated 
immunocytes to become more responsive to secondary exposure to 
immunosuppressants for increased efficacy at a local level. In addition, 
high concentrations must be delivered to the local site to achieve such 
efficacy. This likely models an activated autoimmune disease state in 
tissue such as psoriasis in skin. 
More specifically, data shows that to inhibit an ongoing inflammatory 
immune response, a key factor is that cyclosporine must be delivered 
locally at high concentrations (&gt;10,000 ng/gm or ml) during primary 
topical treatment. Another important item is that prior cyclosporine 
exposure can greatly influence site-specific effects. Thus, more 
efficacious immune modulation at a systemic level predisposes to increased 
efficacy, locally (FIGS. 1 and 4). In addition, immunogenicity at the 
local site decreases during treatment. For example, immunocytes obtained 
from animals following systemic/topical treatment, show increased 
sensitivity to secondary exposure of cyclosporine at a local level. This 
occurs both at a basal level and following antigen activation. Thus, based 
upon pharmacokinetic studies, the dose required to inhibit immune 
responsiveness locally at half maximal effectiveness (Kd) during secondary 
exposure is greatly reduced compared to primary exposure. Additionally, 
these immunocytes also demonstrate antigen specific unresponsiveness 
(tolerance) and therefore the Kd for immunosuppressant cyclosporine 
treatment during secondary antigen exposure is also greatly reduced. In 
practical terms, based upon these data, one concludes that during an 
activated autoimmune response, the efficacy for topical cyclosporine will 
increase following combined systemic/topical treatment and with subsequent 
rounds of local treatment alone. 
Both in vitro and in vivo data confirms that CsA does indeed have potent 
immunosuppressive effects on preactivated cells, as concentrations are 
increased to relatively high values locally (FIGS. 1 and 4). Such levels 
in the systemic circulation would normally be expected to produce organ 
toxicity. However, high levels of CsA can be localized at the tissue site 
undergoing immune activation. Animal experiments show that local CsA is 
efficacious and non-toxic. 
Test Systems 
Topical formulations of cyclosporine, rapamycin, and other 
anti-inflammatory compounds have been successfully developed and tested in 
animal studies. They have been studied for transdermal penetration and 
their ability to effect localized anti-inflammatory responses in the skin. 
Certain principles have been defined, with respect to the vehicle (see 
below), as important and necessary for efficacy. In addition, dose and 
timing requirements have been studied and a critical method has been 
identified for successful treatment. In addition, a novel skin graft 
animal model has been developed to screen anti-inflammatory formulations, 
their efficacy, toxicity, and mechanisms of action. 
Our hypothesis to achieve local efficacy is to deliver high levels of the 
immunosuppressive compound at the site of highest immune cellular 
interactions. Therefore, a depot effect of the active principle in the 
dermal tissues or high concentration gradient would be the desirable 
result. In studies with various formulations, none have generated 
measurable levels of CsA in aqueous transdermal collecting solutions using 
in vitro diffusion chambers. This data includes formulations that have 
clearly demonstrated transdermal CsA levels and efficacy in vivo (FIG. 6). 
FIG. 6 shows that certain classes of carriers, can serve as vehicles for 
transdermal delivery of cyclosporine. Data represented in these examples, 
demonstrates that some carriers can enable the lipophilic cyclosporines to 
effect transdermal delivery through the skin and provide systemic 
therapeutic levels. Vehicle systems that increase cyclosporine's 
lipophilicity in partition coefficient experiments (high apparent 
partition coefficients in octanol/HBSS systems) provide increased 
transdermal delivery in vivo (FIG. 7). An example is ethyl alcohol: 
propylene glycol: glycerine (1:6:3) and CsA (2.5% w/v; formula L.1). 
Vehicle systems that decrease cyclosporine's lipophilicity provide depot 
effects in vivo (FIG. 7). In FIG. 7: L.1 is Ethanol:1,2 
Propanediol:Glycerine (1:6:3); TRS is Diethylene glycol monoethyl ether 
(transcutol); PG is 1,2 Propanediol; LIL is Vegetable oil PEG-6 complex 
(Labrafil); LOL is Labrasol=Glyceryl caprylate/caprate (and) PEG-8 
caprylate/caprate (Labrasol); OLV is Olive oil; D-T is Lanoline: Mineral 
oil: Olive oil (1:1:1); ORL is Oral Sandimmune Liquid; ETH is EtOH 
(Ethanol); and D is Lanoline: Mineral oil: Olive oil:Polysorbate 80 
(1:1:1:0.57). 
With regard to in vitro test systems of skin penetration, due to 
insolubility of the lipophilic active in standard aqueous based collecting 
solutions used for in vitro chamber studies, the value of such analyses is 
questionable. An example of in vitro results from one formulation that is 
known to act transdermally in vivo is shown in Table I. As can be seen, 
CsA is clearly deposited in the skin. The distribution is presented as a 
percent of the total amount of CsA detected in pig skin. 
TABLE I 
______________________________________ 
stratum corneum 11% 
epidermis 75% 
dermis 14% 
chamber solution 0% (undetectable) 
______________________________________ 
Therefore, one of the most important factors in topical immunosuppressive 
therapy is to localize the compound in the area of cellular activity. This 
increases the efficacy of the compound and decreases the toxicity to 
uninvolved organ systems. In vitro diffusion chamber tests on certain 
formulations support the premise that CsA localizes in the skin (stratum 
corneum, epidermis and dermis) with very little penetrating transdermally 
into the aqueous chamber solution. In vivo test results have confirmed our 
hypothesis that such in vitro assay systems are not completely adequate 
for evaluating topical cyclosporine and other lipophilic 
immunosuppressants. Conclusions must be derived in conjunction with in 
vivo test results. In vivo models have been designed that provide superior 
test systems concerning absorption and functional data in these 
situations. The functional results of local immunosuppressive therapy are 
much more relevant to the clinical situation and are detailed below. 
In Vivo Penetration Studies 
Various topical drug formulations have been successfully devised, 
incorporating certain chemical properties within the drug vehicle (see 
below). Certain classes of carriers, or combinations thereof, can serve as 
vehicles for lipophilic immunosuppressants such as cyclosporine which 
enable the active principle to penetrate and localize within the skin 
tissue (see FIGS. 3,8,9). It has been proven that this is one of the key 
factors in achieving local efficacy at the tissue site without evidence of 
systemic delivery or actions. However, other carriers can enable 
lipophilic immunosuppressants such as cyclosporine to effect primarily 
transdermal delivery through the skin and provide systemic therapeutic 
actions (FIGS. 6A, B and C). 
Site-specific immune suppressive drugs can be formulation dependent 
Some formulations are not efficacious. This is congruent with published 
results from other investigators. Surprisingly, however, certain 
formulations have been found extremely efficacious. Certain principles 
have been defined as necessary to achieve site-specific immunosuppression 
by topical cyclosporine. These principles are detailed below. 
Certain classes of carriers, can serve as vehicles for transdermal delivery 
of cyclosporine. Data represented in these examples, shows that some 
carriers can enable the lipophilic cyclosporines to effect transdermal 
delivery through the skin and provide systemic therapeutic levels. Vehicle 
systems that increase cyclosporine's lipophilicity in partition 
coefficient experiments (high apparent partition coefficients in 
octanol/HBSS systems) provide increased transdermal delivery in vivo 
(FIGS. 6-8). Vehicle systems that decrease cyclosporine's lipophilicity 
provide depot effects in vivo (FIGS. 6-8). 
Results were derived from determination of partition coefficients into 
lipophilic/hydrophilic phases with different immunosuppressant vehicle 
solvent systems as follows. One-Octanol was chosen as the hydrophobic 
phase because it is widely believed to mimic biological lipophilic 
environments. RPMI was used as the aqueous phase in order to mimic extra 
cellular fluid. All vehicles were prepared by a standard protocol at 
60.degree. C. Vehicles were spiked with radio-labelled tritiated drug 
solution (Sandoz). 
Fifty ml of 1-octanol and fifty ml of RPMI were added to a fleaker with 
constant stirring. The mixture was then vigorously mixed for two hours at 
4.degree. C. The solution was then removed from the refrigerator and 
placed in a fume hood and allowed to separate for two hours. The 1-octanol 
layer was then removed. To five ml of the 1-octanol saturated RPMI, 500 
.mu.l of tritiated vehicle at 60.degree. C. was added with vortexing. The 
same procedure was also completed with non-labeled vehicle. The tubes were 
then placed in a tube rotator at 17 RPM for 48 hours at room temperature, 
24.degree. C. The samples were then centrifuged for 10 minutes at 
1155.times.g. Three 1 ml samples of the octanol layer of each tube was 
removed and put in a separate 13 ml scintillation vial. About 3 ml of the 
interface was then removed and three one ml samples of the RPMI layer 
drawn from the bottom of the tube were then placed in three separate 
scintillation vials. Thirteen ml of ecoscint were then added to each of 
the scintillation vials and their counts were measured using a liquid 
scintillation counter. 
Functional/Pharmacology Data 
These studies show that one can partition CsA into the skin and inhibit 
functional immune responses (T cell activation and inflammatory reactions) 
at a local level without significant influence on systemic immunity (FIGS. 
9 and 10). Anti-inflammatory efficacy was proven both grossly, 
histopathologically, and immunologically in the presence of locally 
administered CsA. CsA levels were low systemically, and showed relative 
site-specificity in terms of tissue concentration. Interestingly, of the 
CsA that can be measured in the serum and is derived from transdermal 
delivery, it appears to be parent compound (FIG. 8C). An antibody was 
utilized to determine serum CsA concentration which was directed against 
parent compound for the specific assay and another that was directed 
against metabolites for the non-specific assay. The data suggested that 
very little metabolism of the parent compound takes place during 
transdermal delivery. 
Functional Immune Data 
A number of experiments have been devoted to understanding immune 
mechanisms involved in topical cyclosporine's action. In summary, it has 
been shown that localized cyclosporine down regulates the expression of 
both MHC class I and II target antigens compared to placebo treated grafts 
as determined by immunohistochemistry (FIG. 11). This may inhibit antigen 
presentation by APCs, T cell recognition, binding, cellular communication, 
and maturation. Moreover, topical cyclosporine has been shown to inhibit 
either the infiltration or proliferation of CD4+ MHC class II responsive T 
helper cells locally. In addition, data supports the hypothesis that 
systemic antigen specific suppressor cells and/or clonal deletion develops 
following antigen stimulation and local CsA treatment (FIG. 12). This 
latter finding is very important because it demonstrates that efficacious 
immune modulation at the site of activation can have profound beneficial 
influence upon systemic immunity, with reduced side effects. 
Topical drug formulations which combine active agents to synergistically 
attack multiple disease mechanisms at a local site-specific level 
Results above demonstrate that multiple classes of active agents can be 
successfully combined to produce new and extremely potent topical drugs 
(see FIGS. 2 and 13). The liquid pharmaceutical composition used in FIG. 
13A comprised a novel immunosuppressive drug, a steroid or corticosteroid, 
and a carrier consisting of the following: (1) an esterification product 
of natural triglycerides and polyethylene glycol which may be prepared 
according to U.S. Pat. No. 3,288,824; (2) a vegetable oil; and (3) 
ethanol. 
The preferred immunosuppressant is cyclosporine A and the preferred 
corticosteroid is hydrocortisone. The preferred embodiment contains CsA to 
corticosteroid 2.5 to 1 (i.e., range CsA 0.1% to 25%: corticosteroid 0.01% 
to 10%; preferred range 2.5% CsA: 1.0% hydrocortisone). The preferred 
embodiment contains ester to cyclosporine in a weight ratio of about 
10:0.2 to 10 parts by weight; vegetable oil is 35 to 60% of the total 
composition by weight; and ethanol is 1 to 20% of the total composition by 
weight. The steroid is not limited to hydrocortisone. 
For example, a topical oleaginous ointment base comprising the combined 
active ingredients of cyclosporine and an off-patent steroid, 
hydrocortisone, provide significant increased efficacy. In this instance, 
a relatively weak corticosteroid (hydrocortisone) was successfully 
combined with CsA to provide extensive and site-specific graft 
prolongation with topical treatment. However, induction of immune 
unresponsiveness in this situation is critically dependent upon a local 
phase of cyclosporine treatment independent of other immunomodulators. 
Thus, new compounds that combine more than one active principle with 
cyclosporine can be designed to synergistically attack multiple 
inflammatory mechanisms and functions with reduced side-effects. This 
affords the opportunity for developing novel local immunosuppressants 
based topical drugs by combining off-patent active principles and/or other 
promising new agents. 
Examples of other agents that could analogously be combined with novel 
microorganism derived immunosuppressants in a single topical formulation 
in order to potentially enhance efficacy include but are not limited to: 
Betamethasone dipropionate; betamethasone valerate; fluocinolone 
acetonide; triamcinolone acetonide; prednisone; methylprednisolone; 
prednisolone indomethacin; sulindac; ibuprofen; aspirin; naproxen; 
tolmetin; azathioprine; cyclophosphamide; deoxyspergualin; bredinin; 
didemnin B; methotrexate; and thalidomide. Other additional actives and 
potential combinations are listed below. 
cyclosporine-rapamycin synergism 
As discussed above, immune/inflammatory reactions can be inhibited via 
topical use of cyclosporines, alone and in combination with other 
anti-inflammatory agents, and more particularly, with Cyclosporine A alone 
or combined with steroidal anti-inflammatory agents such as hydrocortisone 
to produce synergistic results. However, multiple immunosuppressant agents 
can be combined, particularly with CsA, to produce potent topical 
immunosuppressants. In the instance described above, a relatively weak 
corticosteroid (hydrocortisone) was successfully combined with CsA to 
provide extensive and site-specific graft prolongation. (FIGS. 2, 13A). 
Topical Rapamycin (Alone without combination)--examples of site-specific in 
vitro models 
Rapamycin or CsA alone, provided potent site-specific immune suppression in 
either primary or activated cellular immune responses as in vitro models 
of local immune suppression. Cyclosporine or rapamycin inhibited primary 
inflammatory/immune responses by local application in mixed lymphocyte 
responses (FIGS. 14 A and B). Rapamycin was surprisingly efficacious with 
local application during both late and early inflammatory immune phases. 
Cyclosporine was particularly efficacious locally during the early 
inflammatory immune phase compared to the late phase (FIGS. 14 A and B). 
Examples of site-specific in vivo models alone, and in combination 
Consistent with these in vitro findings, either cyclosporine or rapamycin 
inhibited local inflammatory/immune responses by topical application to 
skin tissue undergoing hypersensitivity responses. This included 
site-specific immune suppression effected by topical use of cyclosporine 
and rapamycin combinations in contact hypersensitivity reactions of skin 
tissue (FIG. 13B). In FIG. 13B, rapamycin, alone, and in combination with 
CsA, provided new potent site-specific topical drugs in a mouse contact 
dermatitis model. Rapamycin (0.01%), cyclosporine (0.1%), and 
rapamycin-cyclosporine (0.01%/0.1%) combined formulations, were applied in 
a trinary drug delivery system consisting of 1,2 propanediol, diethylene 
glycol monoethyl ether, and a glyceryl caprylate/caprate polyethylene 
glycol complex. This vehicle system was surprisingly effective at inducing 
site-specific immune suppression even at these low concentrations of novel 
immune suppressants. Additionally, as discussed previously, a relatively 
weak corticosteroid (hydrocortisone) was successfully combined. Therefore, 
multiple agents can be combined with CsA to produce potent topical 
immunosuppressant drugs. 
Similar to the in vitro data, rapamycin was particularly efficacious during 
the late local inflammatory-immune phase in this latter example, and 
cyclosporine was particularly efficacious during the early local 
inflammatory immune phase. Rapamycin (alone), at concentrations as low as 
0.001%, provided surprising site-specific immune suppression. In addition, 
cyclosporine (0.01%) alone, was surprisingly efficacious using the 
particular delivery system detailed above. Rapamycin in combination with 
CsA (0.001%/0.01%), also provided unexpected site-specific immune 
suppression by topical application in this mouse contact dermatitis model 
using the same delivery system. 
To further elaborate, rapamycin (0.001%), cyclosporine (0.01%), and 
rapamycin-cyclosporine (0.001%/0.01%) combined formulations, were applied 
in a trinary drug delivery system consisting of 1,2 propanediol, 
diethylene glycol monoethyl ether, and a glyceryl caprylate/caprate 
polyethylene glycol complex (6:3:1). Rapamycin derivatives and analogs, 
and other immunophilin binding macrolides and immunosuppressive agents 
shall similarly be effective using these methods and delivery systems. 
Other anti-inflammatory immunosuppressant combinations that can similarly 
be used include but are not limited to the following: DSG and analogs; 
mycophenolic acid derivatives--RS61443; purine and pyramidine blockers; 
brequinar and derivatives; immunophilin binding immunosuppressants; 
macrolide immunosuppressants; FK506 and derivatives; and others. 
Other immunosuppressants (alone) for site-specific effects can include but 
are not limited to the following: DSG and analogs; mycophenolic acid 
derivatives--RS61443; purine and pyramidine blockers; brequinar and 
derivatives; immunophilin binding immunosuppressants; macrolide 
immunosuppressants; FK506 and derivatives; and others. 
Other site-specific applications and compositions can include but are not 
limited to the following: pumps; shields; liposome formulations; 
microemulsion formulations; encapsulations; lipid emulsions; organ 
perfusion; ophthalmic, shampoo, mouthwashes, ear, inhalants, 
suppositories, joints etc.; systemic/site-specific combinations; patch, 
solid and transdermal delivery systems; and formulations for site-specific 
injection as in joints for inflammatory/immune conditions including 
rheumatoid arthritis. 
Localized site-specific efficacy within skin tissue 
Non-polar oleaginous hydrophobic/lipophilic carriers, or combinations 
thereof, can serve as vehicles for novel lipophilic immunosuppressants, 
such as the cyclosporines. In these instances, certain oleaginous carriers 
enable the lipophilic active principle to penetrate the epidermal barrier, 
depot within the tissue in high concentrations, and effect local 
site-specific immunosuppression. It has been proven that such classes of 
carriers are one of the key factors in achieving local efficacy at the 
tissue site without evidence of systemic delivery or actions. Examples of 
such hydrophobic carriers which have been tested and shown to uphold these 
principles include but are not limited to the following or combinations 
thereof: Anhydrous Lanolin; Petrolatum; Vegetable Oils; Mineral Oils; 
Jojoba Oil; and other oils and waxes. 
Transdermal drug delivery through skin tissue 
Most surprisingly and in contradistinction to the results above, broadly 
efficient polar organic solvents, carriers, or combinations thereof, can 
enable novel lipophilic immunosuppressants derived from microorganisms, 
using cyclosporine as a model, to effect transdermal delivery through the 
skin. These solvent systems are effective solubilizers of 
immunosuppressants. In general, solubilizing efficiency of the solvent 
systems, the solubility of the active compound, and the polar nature of 
the organic carriers appears to influence levels of transdermal drug 
delivery. These solvents can be combined to make efficient solvent 
systems, preferably, consisting of binary and trinary systems. 
For example, solvents and carriers demonstrating solubility in, or 
miscibility with a broad range of organic solvents, and even water are 
preferred. Ethoxylates such as diethylene glycol monoethyl ether is a 
preferred lipo- and hydrosoluble solvent. It is miscible with acetone, 
benzene, chloroform, ethanol, and water. Another preferred class of 
solvents are hydrosoluble oils with moderate to high HLB valves. Another 
preferred class of solvent is glycolysed ethoxylated C8/C10 glycerides 
such as glyceryl caprylate/caprate and polyethylene glycol 
caprylate/caprate, also known as Labrasol. This solvent is a hydrosoluble 
oil with a high HLB value of 14 and soluble in ethanol, chloroform, and 
water. Another preferred solvent is propylene glycol which is miscible 
with water, acetone, chloroform, and essential oils. Glycolysed 
ethoxylated glycerides with moderate to high HLB values are also preferred 
co-solvents and carriers including amphipathic esters such as vegetable 
oil polyethylene glycol-6 (PEG) complexes (Labrafils), and similar agents. 
Other preferred solvents and carriers satisfying the physico-chemical 
criteria above include but are not limited to: diethylene glycol diethyl 
ether, dimethylformamide, dimethyl sulfoxide, ethylene glycol monoethyl 
ether, polysorbates, polyethylene glycol, polyoxyethylene esters and 
alcohols. Preferred solvent and co-solvent combinations for transepidermal 
and transdermal delivery are trinary and binary systems. One preferred 
composition is a trinary drug delivery system consisting of 1,2 
propanediol, diethylene glycol monoethyl ether (Transcutol), and a 
glyceryl caprylate/caprate polyethylene glycol complex (Labrasol). Ratios 
of the individual excipient components within this system may vary, but, 
are preferably 6:3:1, respectively. The solvent system can include a 
single component or multiple components and range from 20%-100% but 
preferably is greater than 50% of the total composition. 
Transdermal delivery of cyclosporine in these systems results in systemic 
levels of parent compound, almost exclusively compared to derivatives. In 
these instances, such carriers will localize cyclosporine within the 
tissue in high concentrations, creating a gradient to the systemic 
circulation. If delivered locally at high concentrations, systemic effects 
can result. However, at lower immunosuppressant concentration gradients, 
using this class of solvents, site-specific efficacy can be achieved 
without systemic effects. It has been proven that such classes of carriers 
are one of the key factors in achieving transdermal drug delivery and 
systemic actions. However, unexpectedly, it was found that increasing the 
viscosity of such formulations by thickening agents could retard systemic 
delivery and effects, and even reduce local site-specific efficacy. 
Additional examples of moderately polar solvents, co-solvents and 
transdermal carriers include but are not limited to the following or 
combinations thereof: emulsifying bases and solvents with an amphipathic 
character and moderate hydrophilic to lipophilic balance (HLB) values 
(.gtoreq.3.0) including oil-PEG-6 complexes, polyoxyethylene alcohols, and 
polyoxyethylene fatty acid esters. 
DETAILED DESCRIPTION OF THE INVENTION 
The present invention provides a method and compositions for abrogating 
skin allograft rejection, hypersensitivity reactions, and inflammatory 
reactions. The use of cyclosporine, rapamycin, or combinations of 
immunosuppressant and other anti-inflammatory agents prolongs the survival 
of experimental skin allografts, and/or inhibits contact hypersensitivity 
inflammatory/immune reactions by delivering the drug to the target tissue 
and increasing efficacy. Systemic therapy alone can be excessively 
nephrotoxic, hepatotoxic and neurotoxic, and a concomitant increase in 
infections may pose a significant problem. Use of the present invention, 
however, circumvents these difficulties and provides a treatment 
methodology which effectively emphasizes the positive attributes of 
cyclosporine, rapamycin, and other immunosuppressants while minimizing the 
detrimental side effects. 
EXAMPLES 
Topical applications and compositions--steroid synergism 
A dual skin allograft model was developed to evaluate mechanisms of site 
specific immunosuppression with topical immunosuppressants. It was 
hypothesized that synergistic efficacy could be induced by site specific 
immunomodulation via antigen-specific mechanisms with topical cyclosporine 
(CsA) in combination with topical corticosteroids (CS) acting on 
antigen-independent, nonspecific anti-inflammatory mechanisms. The 
experiment was designed with a vehicle system that provided 
less-than-optimal transepidermal delivery and graft prolongation in order 
to test for potential synergism. Three different protocols were evaluated: 
1) Topical CsA (2.5%) at the induction phase through the entire rejection 
process; 2) Combined topical CsA/hydrocortisone (HC; 2.5%/1.0%) at the 
induction phase through the entire rejection process; and 3) Topical CsA 
during the immune induction phase and subsequent addition of combined 
CsA/HC at initiation of the maintenance phase to full rejection. Topical 
CsA provided moderate graft prolongation and disparity (FIG. 2). Mean 
survival times increased slightly with combined topical CsA/HC in 
comparison to CsA alone, but, did not provide a significant synergism 
(FIG. 2). However, topical CsA during immune response induction or the 
antigen-dependent phase with subsequent suppression of antigen-independent 
inflammation by topical CsA/HC provided dramatic synergism with optimal 
efficacy and disparity (FIG. 2). Therefore, it was concluded that a local 
and beneficial immuno-regulatory mechanism was induced by topical CsA 
during the early immune induction phase which was sensitive to 
corticosteroids and inhibited during concomitant corticosteroid therapy. 
However, addition of a corticosteroid during the maintenance phase was 
necessary for this beneficial mechanism to be expressed. 
Cyclosporine and corticosteroids (CS) are widely used immunosuppressive 
drugs in transplantation and autoimmunity (1-6). However, controversy 
exists with respect to achieving effective combinational therapy with 
these drugs for modulating an active on-going immune response. 
There exists experimental evidence that both the induction of the immune 
response and the effector mechanisms during cellular responses are 
regulated locally at the site of antigenic challenge (1,2,7). In fact, a 
dual skin allograft model developed in our laboratory provided evidence 
that site-specific immunosuppression with CsA could modulate immune 
responsiveness locally (1,2). Our studies established an ongoing 
Ag-specific driven immune response which is CsA sensitive. Depending on 
the formulation and CsA concentration delivered locally, this phase can be 
followed by effector cell generation. Eventually, this effector cell 
generation may penetrate and escape the CsA coverage leading to the 
recruitment of non-specific inflammatory components in an environment rich 
with various mediators. These later phase Ag-independent, lymphokine 
driven events tend to be less sensitive to the suppressive effects of CsA 
(1,2,8,9), since CsA is primarily known to work by affecting lymphocyte 
activation and lymphokine release (9,10). Thus, a localized site-specific 
modulation of antigen-dependent immune response induction can be 
hypothesized to be optimally achieved early on. It is also very 
conceivable that CsA may play an active role at inducing or facilitating 
the development of local immuno-regulatory mechanisms as well. It has been 
documented that CsA spares suppressor networks and facilitates regulatory 
events systemically (10). 
Localized corticosteroids would theoretically make a good adjunct as a 
combinational therapy with topical CsA since they act as potent 
anti-inflammatory agents and non selective immunosuppressants (11,12) that 
may inhibit antigen-independent inflammatory reactions. Corticosteroids 
are believed to work by stabilization of cellular membranes, inhibition of 
cellular maturation, proliferation, and migration (4,11-14). 
We hypothesize that CsA induces and/or facilitates site-specific 
immunosuppressive mechanisms which could then act synergistically with 
corticosteroid Ag-independent inflammatory suppression. However, the 
non-specific immunosuppressive nature of corticosteroids may potentially 
abrogate putative localized site-specific regulatory mechanisms 
established under CsA therapy. Therefore, a corticosteroid addition may be 
detrimental during certain cellular phasic events occurring along the 
course of immunomodulation. Thus, a timing factor in the addition of a 
corticosteroid may be crucial in the hypothesized synergistic effect of 
both immunosuppressive drugs. Indeed, activated inflammatory immune 
responses have a higher signal sensitivity, different pattern of 
re-circulation, lower T cell dependence, and a lower antigen concentration 
requirement (15-20). As stated previously, CsA may be less effective in 
overcoming a preactivated response (1,2,8), but inclusion of a 
non-specific, membrane-stabilizing agent like a corticosteroid may provide 
synergism and a potent combinational site-specific immunosuppressive drug. 
The success of CsA/CS therapy would depend on the existence and 
preservation of CsA's regulatory characteristics and the ability of 
corticosteroids to function in conjunction with CsA to modulate 
pre-activated responses. 
Animals 
In this investigation, male Lewis x Brown Norway F.sub.1 rats (LBN, 
RT1.sup.1+N) were used as skin donors to male Lewis (LEW RT1.sub.1) 
recipients. The animals weighing around 350 grams were maintained in a 
temperature controlled environment at the University of California, Irvine 
vivarium facilities for the duration of the experiments. 
Dual Skin Allograft Model 
Split thickness donor skin, 0.015 inches in thickness, was taken from the 
dorsal side of LBN rats with a Gibson Ross dermatome (Thackery 
Instruments, England). Each graft (3.times.4 cm.sup.2) was transplanted 
onto the dorsal side of the recipient using 3-0 sutures and stay sutures 
to secure the grafts. Each graft was monitored daily for erythema, hair 
growth, eschar, exudate and scabs to determine graft status. When fifty 
percent of the graft was graded to be necrotic tissue, then the allograft 
was determined to be at a peak of rejection (50% rejection). First sign of 
rejection was determined based on the initial observation of erythema, 
continuous hair loss, flakiness and/or scabs without reverting back to 
previous conditions (1st sign). Full rejection was characterized by 
complete necrosis of the allograft skin tissue and establishment of scar 
tissue (full rejection). 
Experimental Protocol 
Three different protocols were employed to study the mechanisms of 
site-specific immunosuppression with CsA-HC combinational therapy. All 
groups received 10 days of systemic CsA (Sandoz oral solution, Sandoz, 
USA) post-transplantation. Topical (2.5%) CsA was made in a oleaginous 
base formulation that provided moderate local efficacy (EPIX, Santa Ana, 
Calif., see below). In regimen 1, 5 mg/Kg/day of CsA was applied topically 
following the 10 days of systemic CsA treatment until full rejection as 
described previously. Topical application of a mixture of 2.5% CsA and 
1.0% HC following the 10 days systemic treatment was employed for regimen 
2. For regimen 3, topical application of 2.5% CsA alone was applied until 
initial signs of rejection, then was switched to the combinational mixture 
formulation until full rejection. 
Statistical Analysis 
Appropriate use of the unpaired and paired Student-T test was employed for 
all evaluation of data. Survival data was converted to a logarithmic 
transformation to correct for skewness. P values were considered 
significant if they were less than 0.05. 
CsA provided prolongation of skin allografts in a site-specific manner when 
applied topically using the dual skin allograft model. In FIG. 2 the 
survival of placebo treated skin allografts and disparity in survival 
between experimental and placebo treated grafts are displayed. A base 
formulation was utilized that provided moderate efficacy with CsA in 
comparison to previously published formulations (1,2), but was compatible 
with HC formulation and addition. For each group, a significant disparity 
between the experimental graft and placebo graft was obtained (p&lt;0.05). 
The experimental graft appeared normal for the duration of the 
prolongation phase with full hair growth and normal skin texture. Three 
different protocols were evaluated: 1) Topical CsA (2.5%) at the induction 
phase through the entire rejection process; 2) Combined topical CsA/HC 
(2.5%/1.0%) at the induction phase through the entire rejection process; 
and 3) Topical CsA during the immune induction phase and subsequent 
addition of combined CsA/HC at initiation of the maintenance phase to full 
rejection. Topical CsA provided moderate graft prolongation and disparity 
(FIG. 2). Mean survival times increased slightly with combined topical 
CsA/HC in comparison to CsA alone, but, did not provide significant 
synergism (FIG. 2). However, topical CsA during immune response induction 
or the antigen-dependent phase with subsequent suppression of 
antigen-independent inflammation by topical CsA/HC provided dramatic 
synergism with optimal efficacy and disparity (FIG. 2). 
Addition of topical HC in combination with topical CsA at first sign of 
rejection to an allograft previously treated with topical CsA alone 
provided the synergistic combinational effect. Prior to the first sign of 
rejection, the experimental graft of group was similar to the results of 
groups 1 and 2. However, addition of corticosteroid in combination with 
CsA after initial signs of rejection reversed the erythema and hair loss 
associated with rejection in all of the animals in this group. The graft 
returned to good health with full hair growth in 4 out of 5 animals. 
Notice that the disparity in group 3 was approximately 3.times. more than 
groups 1 or 2. The synergistic effects were expressed only during the 
maintenance phase of CsA/HC combined treatment during the overlapping of 
Ag-dependent and Ag-independent mechanisms. 
Systemic profiles of serum CsA yielded a predictable trend in placebo 
treated skin allograft survival (FIG. 3). Ten day subcutaneous treatment 
reached a peak of 1,700 ng/ml at day 11. Subtherapeutic levels were 
reached and maintained by day 25. Placebo grafts rejected shortly 
thereafter, as expected. 
These results demonstrate that topical CsA is effective at locally 
modulating early immune events during the induction phase which can be 
considered primarily an antigen-dependent process. CS can be considered to 
primarily inhibit antigen-independent, nonspecific inflammatory reactions. 
Thus CS may be most effective during recruitment of nonspecific 
inflammatory components which are less sensitive to CsA. The simultaneous 
addition of CsA and CS in topical formulations would theoretically enhance 
immunomodulation since concomitant inhibition of antigen-dependent and 
antigen-independent responses by these agents, respectively, would be 
active (Group II). However, significantly enhanced immunomodulation was 
not observed in this case. Based on the mechanisms of action of both 
drugs, results suggest that CS may suppress the beneficial expression of 
CsA induced, immunoregulatory mechanisms. However, these beneficial 
mechanisms were only expressed with the subsequent addition of 
antigen-independent inflammatory cascade suppression by CS in combination 
with CsA (Group 3). 
It has been established that corticosteroids down-regulate Major 
Histocompatibility Complex (MHC) class II expression and destabilize 
membranes of Antigen Presenting Cell (APC) and/or immunocompetent cells 
(20). Since antigen recognition has been shown to be important for CsA's 
mechanism of action and immunosuppressive efficacy (9,10), CS may inhibit 
CsA immunosuppression by interfering with these early activation events. 
CS may also abrogate on-going mechanisms of readaptation within the graft. 
It has been demonstrated the CsA spares or even facilitates suppressor 
T-cell expression (10). But, the development of, and even the effect of, 
the suppressor cell network could possibly be abrogated locally by CS 
addition as shown in other studies (21-23). Also, peripheral selection of 
the T cell repertoire is dependent on T cell receptor (TCR)-ligand 
interactions (24). If CS is applied at the beginning of the Ag-dependent 
immune response induction phase, then peripheral selection of local T 
cells could be impaired, since CS destabilizes membranes and down 
regulates receptor interactions (11-14). Therefore, CsA induced clonal 
deletion mechanisms may not be developed under concomitant CS coverage. 
However, the deletion of allo-responsive cells during early CsA sensitive 
phases could act synergistically with Ag-independent suppression when 
non-specific inflammatory elements are recruited (initiation of the 
rejection phase). 
A decrease in alloresponsive cells would result in low mediator release and 
hence low recruitment of non-specific inflammatory elements. Studies 
performed using infiltrating sponge matrix allograft models demonstrate at 
the peak of infiltration only 0.2% of the cells were Ag-specific T 
cytotoxic lymphocytes (25,26). Thus, this implies that the other 99.8% are 
non-specific inflammatory cells responding to production of immune 
mediators by the initially small numbers of antigen-specific infiltrating 
cells. This may also explain the synergistic effect observed in our 
experiments. 
Since the simultaneous administration of CsA and HC during the induction 
phase did not provide synergistic site specific immunomodulation, we can 
conclude that the development of regulatory mechanisms induced by CsA at 
this time are steroid sensitive. However, once these regulatory elements 
were established, their effects were not steroid sensitive. The results of 
this study, therefore, suggests that a topical CsA/HC combinational drug 
or possibly even systemic CsA/HC therapy can be optimally employed with 
proper timing. This would allow both CsA and HC to result in their most 
potent effect synergistically on selective cellular and phasic events 
occurring along the course of antigen-dependent, immune responsiveness, 
and nonspecific inflammatory responsiveness. Therefore, the timing of 
corticosteroid addition during immune response induction in combination 
with CsA should be considered carefully since it may potentially negate 
the beneficial regulatory mechanisms at critical phases triggered by CsA. 
Studies are currently in progress using immunohistochemical staining and 
cell trafficking techniques to further study these mechanisms of action. 
The application of CS at the first sign of rejection has been used 
systemically for salvage of CsA treated kidney, liver, and heart grafts in 
clinical situations (27-31). Similarly, use of CS site-specifically or 
topically, in combination with CsA has surprising benefits. The optimal 
therapeutic protocol for combinational therapy of CsA and CS would be 
suggested to be when nonspecific inflammatory events are initiated 
following the induction of beneficial CsA immunomodulatory mechanisms. 
In conclusion, these results provide further support of our previous 
findings that local site-specific or systemic efficacy via topical 
delivery to the skin can be dependent upon carrier composition with 
particular emphasis on solvent efficiency, the hydrophilic/lipophilic 
nature of the vehicle, viscosity, active principle solubilization, and 
concentration. 
Topical formulations of cyclosporine and other anti-inflammatory compounds 
have been successfully developed and tested in animal studies. They have 
been studied for transdermal penetration and their ability to effect 
localized anti-inflammatory responses in the skin. Certain principles have 
been defined, with respect to the vehicle, as important and necessary for 
efficacy. In addition, dose and timing requirements have been studied and 
a critical method has been identified for successful treatment. In 
addition, a novel animal model has been developed to screen 
anti-inflammatory formulations, their efficacy, toxicity, and mechanisms 
of action. Systemic application is necessary to modulate the immune 
response in accord with the known mechanisms of actions of cyclosporine. 
In addition, as wound healing is expedited with systemic administration, a 
nidus of inflammation is therefore avoided. In conclusion, it is believed 
that these findings are directly transferable to other inflammatory 
reactions including autoimmune diseases of the skin in the clinical realm. 
The solubility of the compound and polarity of the carriers appears to 
influence levels of transdermal drug delivery. Certain non-polar 
oleaginous hydrophobic/lipophilic carriers, in these instances, enable the 
lipophilic active principle to penetrate the skin barrier, depot within 
the tissue in high concentrations, and effect local site-specific 
immunosuppression. Carriers with an increased polar nature and that are 
broadly efficient solvents enhance transdermal penetration and therefore, 
local and systemic effects. It has been proven that such classes of 
carriers are one of the key factors in achieving transdermal drug delivery 
and systemic actions. Different formulations can be easily devised to 
produce creams or ointments which may prove advantageous. 
It will be understood that the scope of this invention is not limited by 
the above-described preferred embodiment. It will be understood that 
various changes and modifications can be made therein without departing 
from the spirit and scope of the invention, which are defined by the 
following claims. 
REFERENCES 
1. Black K.,s. Hewitt C. W., C. L. C. Chau, and L. Pizzo. "Transdermal 
Application of Cyclosporine Prolongs Skin Allograft Survival." Transplant. 
Proc. 1988; 20(2):660-662. 
2. Black K. S., Nguyen D. K., Procter C. M., Patel M. P., Hewitt C. W. 
"Site-specific Suppression of Cell-Mediated Immunity by Cyclosporine." J. 
Invest. Derm. 1990; 94(5):644-8 
3. Griffiths C. E. M, Powles A. V., Baker B. S. and Valdimarsson H. 
"Combination of Cyclosporine A and Topical Corticosteroid in the Treatment 
of Psoriasis." Transplant. Proc. 1988; 20(3):50-52. 
4. Toyry S., Fraki J. and Tammi R. "Mast Cell Density in Psoriatic Skin. 
The Effect of PUVA and Corticosteroid Therapy." Arch. Dermatology 1988; 
280: 282-285. 
5. Gorensek M. J., Stewart R. W., Keys T. F., McHenry M. C., Longworth D. 
L., Rehm S. J., Babiak T. "Decreased Infections In Cardiac Transplant 
Recipients, on Cyclosporine With Reduced Corticosteroid Use." Clev. Clin. 
J. Med. 1989; 56(7): 690-695. 
6. Tamura F., Vogelsang G. B., Reitz B. A., Baumgartner W. A., and 
Herskowitz A. "Combination Thalidomide and Cyclosporine for Cardiac 
Allograft Rejection." Transplantation 1990; 49(1): 20-25. 
7. Hayry P. and Willebrand E. V. "The influence of the Pattern of 
Inflammation and Administration of Steroids on Class II MHC Antigen 
Expression in Renal Transplants." Transplantation 1986; 42:358-363. 
8. Hopt U. J., Erath F., Schareck W., Greger B., Mellert J. "Effect of 
Cyclosporine A on Local Inflammation in Rejecting Allografts." Tranplant. 
Proc. 1988; 20(2):163-169. 
9. Kahan B. D. "Cyclosporine: The Agent and Its Actions." Transplant. Proc. 
1985; 17(4): 5-18. 
10. Hess A. D., Tutschka P. J. "Effect of Cyclosporine A on Human 
Lymphocyte Response In Vitro." J. Immunonol. 1980; 124(6): 2601-2608. 
11. Boss P. S., Jolley W. B. and Ainsworth E. J. "Mechanisms of Action of 
Topically Applied Triamcinolone Acetonide in Prolonging Skin Allograft 
Survival Time." Transplant. Proc. 1988; 15(1): 17-21. 
12. Ashworth J., Booker J. and Breathnach S. M. "Effects of Topical 
Corticosteroid Therapy on Langerhans Cell Antigen Presenting Function in 
Human Skin." Dermatology 1988; 118:457-470. 
13. Takeda K., Arase S. and Takahashi S. "Side Effects of Topical 
Corticosteroids and Their Prevention." Drugs 1988; 36:15-23. 
14. Topert M. "Perspectives in Corticosteroid Research." Drugs 1988; 
36:1-8. 
15. Sanders, M. E., Makgoba, M. W., Sharrow, S. O., Stephany, D., Springer, 
T. A., Young, H. A. and Shaw, S. "Human Memory T Lymphocytes Express 
Increased Levels of Three Cell Adhesion Molecules (LFA-3, CD2, and LFA-1) 
and Three Other Molecules (UCHL-1, CDw29, and Pgp-1) and Have Enhanced 
IFN-Gamma Production." J. Immunol. 1988 140:1401-7. 
16. Budd, R. C., Cerottini, J. C., and MacDonald, H. R. "Selectively 
Increased Production of Interferon by Subsets of Lyt-2+ and L3T4+ T Cells 
Identified by Expression of Pgp-1. " J. Immunol. 1987; 138:3583-86. 
17. Arthur, R. P., Mason, D. "T Cells That Help B Cell Responses to Soluble 
Antigen Are Distinguishedable From Those Producing Interleukin-2 on 
Mitogenic or Allogenic Stimulation." J. Exp. Med. 1986; 163:774-86. 
18. Swain, S. L., Weinberg, A. D., English M. "CD4+ T Cell Subsets. 
Lymphokine Secretion of Memory Cells and of Effector Cells That Develop 
From Precursors In Vitro." J. Immunol. 1990; 144:1788-99. 
19. Lee, W. T., Yin, X. M., Vitetta, E. S. "Analysis of Murine CD45 R High 
and CD45 Low CD4+ T cells." J. Immunol. 1990; 144:3288-95. 
20. Mellert, J., Hopt, U. T., Erath, F., Holzer, H. "Differential Effects 
of Azathioprine (Aza), Cyclosporine A (CsA) and Dexamethaxone (Dexa) on 
Lymphokine Mediated Inflammation in Rejecting Allografts." Transplant. 
Proc. 1989; 21: 98-9. 
21. Ikeda T, Urchihara M, Daiguji Y, Hasumura Y, and Takeuchi J. 
"Immunological Mechanisms of Corticosteroid Therapy in Chronic Active 
Hepatitis: Analysis of Peripheral Blood Suppressor T-cell and Interleukin 
2 Activities." Clin. Immunol. Immunopathol. 1989; 53(2 pt 1):192-201. 
22. Ikeda T. Daiguji Y. Hasumura Y. and Takeuchi J. "In Vitro Effect of 
Prednisolone on Peripheral Blood Suppressor T Cell Activity in Patients 
With Alcoholic Hepatitis." Clin. Immunol. Immunopathol. 1989; 53:225-32. 
23. Highet A. B. and Ruben Ln. "Corticosteroid Regulation of IL-1 
Production May Be Responsible for Deficient Immune Suppressor Function 
During the Metamorphosis of Xenopus Laevis, the South African Clawed 
Toad." Immunnol. Pharm. 1987 13:149-55. 
24. Rocha B. and Boehmer H. V. "Peripheral Selection of the T Cell 
Repertoire. Science 1991; 251:1225-28. 
25. Orosz C. G., Zinn N. E., Sirinek L., Ferguson R. M. "In Vivo Mechanisms 
of Alloreactivity. Frequency of Donor Reactive Cytotoxic T Lymphocytes in 
Sponge-Matrix Allografts." Transplantation 1986; 41: 75-83. 
26. Orosz C. G., Zinn N. E., Sirinek L., Ferguson R. M. "In Vivo Mechanisms 
of Alloreactivity. Allospecificity of Cytotoxic T Lymphocytes in Sponge 
Matrix Allografts as Determined by Limiting Dilution Analysis." 
Transplantation 1986; 41(1): 84-92. 
27. Halasz N. A., Gamboa E. A., Ward D. M., Steiner R. W., and Bronsther. 
"Kidney Transplantation in the CsA Era." Arch. Surg. 1987; 122:1001-1004. 
28. Novick A. C., Ho-Hsieh H., Steinmuller D., Streem S. B., Cunningham R. 
J., Steinhilber D., Coormastic M., and Buszta C. "Detrimental Effect of 
Cyclosporine on Initial Function of Cadaver Renal Allografts Following 
Extended Preservation." Transplantation 1986; 42:154-158. 
29. Veremis S. A., Maddux M. S., Pollak R., Kline S. S., and Mozes M. F. 
"Alternative Antirejection Treatment With Steroids or Antilymphoblast 
Gobulin in Renal Transplant Patients Receiving Cyclosporine." Tranplant. 
Proc. 1987; 19:1893-1895. 
30. Tilney N. L., Milford E. L., Araujo J. L., Strom T. B., Carpenter C. B. 
Kirkman R. L. "Experience with Cyclosporine and Steroids in Clinical Renal 
Transplantation. Ann. Surg. 1984; 200:605-613. 
31. Najarian J. S., Fryd D. S., Strand M., Canafax D. M., Ascher N. L., 
Payne W. D., Simmons R., Sutherland D. E. R. Ann. Surg. 1985; 201:142-157. 
It will be understood that the scope of this invention is not limited by 
the design of the above described preferred embodiment. While the 
invention is described and taught using the preferred embodiment, it will 
be understood that various changes and modifications can be made therein 
without departing from the spirit and scope of the invention, which are 
defined by the following claims.