Concanavalin A dimers as therapeutic agents

Homogeneous dimeric forms of concanavalin A, including the dimer form of native concanavalin A and dimeric derivatives of concanavalin A, are effective immunoregulatory agents with a good balance of high activity and low toxicity. Immunoregulatory pharmaceutical materials composed of one or a mixture of such isolated dimeric materials are disclosed as are methods of inducing immunoregulation in mammals by administering to such mammals an effective immunoregulatory amount of such pharmaceutical materials.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention is in the field of pharmaceutical chemistry and 
therapeutics. More particularly, it relates to concanavalin A materials 
having immunoregulatory activity and their use in applications where that 
activity is called for. 
2. Prior Art 
Concanavalin A (CA) is a well characterized plant lectin that reacts with 
numerous cell types and various molecules of the mammalian immune system. 
The cummulative effect of these reactions is often unpredictable, since CA 
has been shown both to stimulate and to suppress the immune system in in 
vitro and in vivo tests. Further complicating an understanding of these 
diverse biological effects is CA's molecular heterogeneity. Solutions of 
CA are mixtures of varying proportions of multimolecular aggregates, 
tetramers, dimers, monomers and free and associated monomer fragments. The 
exact composition of this mixture is dependent upon the preparation scheme 
and the type of handling the particular sample has received. H. Markowitz 
and colleagues reported in SCIENCE, 163, 476, that the CA mixture provided 
immunosuppressive activity in grafts in mice. R. Weil, III and colleagues 
reported at TRANSPLANTATION, 17, No. 6 600, (1974) the use of CA to 
increase rat heart allograft survival. 
A variety of CA derivatives are described in the literature. These include 
succinylated CA, or SCA; acetylated CA or ACA; cross-linked CA; hybrid CA; 
and the reaction product of CA with maleic anhydride, etc. With any of 
these materials, there is a need to provide reprodcibility in composition 
and a need to optimize the desired activity while minimizing side effects 
and toxicity. 
STATEMENT OF THE INVENTION 
It has now been found that homogeneous dimeric forms of concanavalin A, 
including the dimer form of native concanavalin A and dimeric derivatives 
of concanavalin A, are effective immunoregulatory agents with a good 
balance of high activity and low toxicity. 
In one aspect this invention provides immunoregulatory pharmaceutical 
materials composed of one or a mixture of such isolated dimeric materials. 
In another aspect this invention provides methods of inducing 
immunoregulation in mammals by administering to such mammals an effective 
immunoregulatory amount of such pharmaceutical materials. 
In a preferred aspect, the invention employs this immunoregulation to treat 
transplant or graft rejection or immune disfunction. 
In another aspect, this invention can provide a treated donor organ or 
tissue for transplantation comprising the organ or tissue in combination 
with the dimeric material. 
DETAILED DESCRIPTION OF THE INVENTION 
The Dimeric Concanavalin A 
The present invention employs dimers of concanavalin A. These materials are 
referred to generically as CAD, or Concanavalin A Dimers. One form of 
dimeric CA is the native dimer having a molecular weight of about 
52-53,000. This material can be obtained by adjusting the pH of a purified 
solution of CA to below about 6.0, such as in the range of pH 4 to pH 6, 
thereby disassociating any CA tetramer or higher aggregates into the 
desired "dimer" form. Such solutions are stable at pH's up to about 6.3 at 
temperatures of from below freezing to about 30.degree. C. Preferred 
solutions of the native dimer have pH's of form about 4.5 to about 6.0 and 
are stored for long periods at temperatures below about 10.degree. C. 
These native dimer materials are characterized by containing at least 
about 80% of their total CA as the dimer form. These materials and methods 
for their preparation are described in Huet, BIOCHEM. 59 627-632 (1975) 
and McKenzie, et al., BIOHEM. BIOPHYS. ACTA. 263, 283-293 (1972). A 
preparation of this native dimer material which is preferred because of 
the pure and exceptionally stable product which it provides is described 
by A. Sophianopoulos, et al in PREP. BIOCHEM., 11 (4), 413-435. These 
papers describe in more detail the factors which favor dimer in a solution 
of CA. These papers are incorporated herein for reference. 
The other "dimeric" materials useful in this invention are 
chemically-modified CA's which have a dimeric structure. These materials 
include the reaction product of CA with succinic anhydride to yield a 
succinylated CA or "SCA", and the reaction product of CA with acetic 
anhydride to yield acetylated CA or "ACA" and the reaction product of CA 
with maleic anhydride to yield maleited CA or MCA. Typical preparations of 
these materials are provided in the paper of Gunther, G. R., et al., PROC. 
NAT. ACAD. SCI., USA, 70, No. 4, 1012-1016 (April 1973) which is 
incorporated herein by reference. Additional representative dimer 
materials include the hybrid dimers made up as a hybrid of native CA 
"monomer" and a SCA, ACA or the like "monomer." Such materials are 
described in Fraser, et al. J. BIOL. CHEM., 251, No. 15, 4622-4628 (Aug. 
10, 1976), which is incorporated herein by reference. An additional group 
of useful dimers are the "cross-linked dimers" formed by reacting CA with 
SCA, ACA or MCA in the presence of a bifunctional coupling reagent such as 
a diisocyanate, in particular a lower alkaline diisocyanate such as 
hexamethylene diisocyanate. Such materials are described in Wang. et al. 
J. BIOL CHEM., 253, No. 9, 3000-3007, (May 10, 1978) which also is 
incorporated herein by reference. 
All of these materials are characterized by comprising two of the 
saccharide-binding subunits, each of molecular weight of about 26,000 so 
as to give rise to a dimer molecular weight of about 52,000-53,000. 
The invention is not limited to the particular dimeric materials just 
related and can employ other chemically modified CA substrates so long as 
the overall structure of the predominant material (i.e. 60% or more, 
preferably 80% or more, and more preferably 90% or more of the total CA) 
is of a dimeric structure. Mixtures of two or more dimeric materials can 
be used, if desired. 
Pharmaceutical Preparations 
The dimeric CA material is formulated into a pharmaceutical preparation, 
generally for parenteral administration (i.e. I.V. or intramuscular 
injection) but also for oral administration. In parenteral administration, 
the preparation can be administered I.V. or I.M. over a prolonged period 
or by periodic injections. 
Suitable preparations are composed of one or more of the dimers in 
association with a pharmaceutically acceptable carrier. The book, 
REMINGTON'S PHARMACEUTICAL SCIENCES, 15th Ed., E. W. Martin, (Mack Publ. 
Co., 1975), discloses typical carriers and methods of preparation. This 
disclosure is incorporated herein by reference. Typical carriers include 
injectable saline, injectable water and injectable solutions, with or 
without buffers, isotonicity agents and the like. 
The injectable compositions contain from about 0.005% to about 20% of the 
dimer, preferably from about 0.01% to about 10% of the dimer, and more 
preferably 0.1% to 5%. Oral compositions can vary over a wider range so 
long as the volume of composition needed to deliver an effective dose is 
reasonable for a patient to consume. 
The materials of this invention find use as immunomodulators. They can thus 
be used to ameliorate the effects of undesired immune system reaction such 
as observed in organ and tissue graft rejection as well as to treat immune 
disfunction conditions such as autoimmune disease states--i.e. lupus, 
diabetes, arthritis, ulcerative colitis, acquired immune deficiency 
syndrome, multiple sclerosis and the like. 
The dimers and compositions containing them can be administered in a 
variety of ways. In tissue and organ rejection settings they can serve to 
prevent rejection. This can be carried out by treatment of the organ or 
tissue prior to implantation in the recipient or by administering the 
dimers to the recipient prior to concurrent with or and/or after the 
transplantation. To treat rejection episodes, administration of the dimer 
can be initiated or augmented at the time rejection is suspected or 
diagnosed. 
These treatments can be carried out with the CAD alone or accompanied by 
other therapeutic strategies such as immunoregulatory drugs for example 
steroids, antimetabolites, cyclosporines, lymphokines or other natural 
products or the like. In addition, these materials can work with donor 
antigens in the form of blood, blood components, particulated blood 
antigens and the like, if desired. 
In autoimmune disease settings, the CAD materials serve to treat the 
disease caused by autoimmunity by regulating the immune process. This can 
be carried out by subjecting the patient to a regimen of treatment 
consistent with the course of the disease. The treatment can halt the 
progression of the disease or reverse damage already done by the disease. 
As in the case of tissue or organ rejection, treatment, the CAD can be 
used alone or in conjunction with other therapeutic strategies. 
As will be apparent from the above, the CAD materials can be administered 
over a wide range of schedules and doses. In the case of graft or 
transplant rejection, dosage can begin prior to or simultaneous with or 
after the graft or transplant is carried out. Typically, the treatment can 
begin from weeks before to a week after the graft or transplant and 
continue for up to a year or more up to the lifetime of the patient. 
In the case of the immune disfunction states, typical courses of treatment 
can run from the time the disease is diagnosed or suspected and is 
continued until the disease is arrested or reversed. Such times can range 
from a single dosing to virtual lifetime treatment. 
Typical dose levels are from 0.1 mg/kg/day to 500 mg/kg/day with dose 
levels of from 0.25 mg/kg/day to 200 mg/kg/day being preferred and with 
dose levels of from 0.50 mg/kg/day to 100 mg/kg/day being more preferred. 
This invention will be further described by reference to the following 
examples. These are provided solely to illustrate modes of practicing the 
invention and are not to be construed as limiting the scope of the 
invention which is, instead, defined by the appended claims.

EXAMPLE 1 
This example illustrates the ability of a therapeutic composition based on 
the CA dimer, succinylated CA, to reduce graft and transplant rejection in 
a mammal animal model. The compositions of the invention prolong survival 
of the treated animals after heart transplantation. 
A composition of the invention is prepared from SCA. The SCA is purchased 
as lyophilized powder from Vector Labs, dissolved to 10 mg/ml in 0.9%w 
normal saline and stored at 4.degree. C. Analysis of the SCA (as well as 
native CA purchased from the same source and made up into an identical 
strength solution) by isoelectric focusing on preformed polyacrylamide gel 
plates shows that the SCA migrates as a single band whereas CA is composed 
of four components migrating with pIs near neutrality. These results 
indicate that the SCA employed is composed essentially completely of the 
desired dimer. 
Male Lewis (LEW, RT1.sup.1), Brown Norway (BN, RT1.sup.n) and 
Lewis.times.Brown Norway F.sub.1, hybrid (LBN) rats weighing between 
220-350 gm are obtained from Charles Rivers or Harlan Sprague Dawley. 
Donor whole hearts are transplanted by primary vascular anastomosis in the 
heterotopic (abdominal) position. LEW recipients of LBN or BN hearts are 
treated I.V. with either saline (control) or SCA beginning two days before 
surgery and continued on a daily basis until the day of rejection. 
Hearts are palpitated daily for the presence of contractile activity. The 
day on which all contractile activity stopped is defined as the day of 
rejection. 
The in vivo activities of SCA is evaluated by determining this dimer's 
ability to prevent cardiac allograft rejection. LBN hearts in untreated 
LEW rats survived for 2.times.8, 9, 12, 14 and 15 days. SCA treatment at 
doses of 3 mg/kg and 15 mg/kg cause grafts to survive 12 and 13 days and 
20, 25, 29, and 34 days, respectively. 
None of the recipients treated with SCA at any dose shown any signs of 
overt, clincial toxicity at any time. After three weeks of treatment with 
15 mg/kg of SCA, the recipients' total white blood cell count is normal. A 
dose of 6 mg/kg of CA causes a 40% reduction in the WBC during the third 
week of treatment. 
EXAMPLE 2 
The materials of this invention are used to control rejection in the clinic 
in the following way. A living related or cadaver kidney donor is 
identified. In the case of the living related donor, blood from the living 
related donor is transfused into the recipient weekly for six weeks prior 
to transplantation. In the case of the cadaver donor, ABO compatible 
random blood transfusions are given according to a similar schedule. 
During this time CAD (for example 10 mg for SCA/kg/day, I.V.) optionally 
with other immunosuppressive strategies (e.g., drugs like prednisone, or 
X-irradiation) is also given. Six days I.V. (in the case of the living 
related donor transplant, or 12 hours (in the case of the cadaver donor 
transplant) before surgery, CAD is given to the prospective recipient at a 
dose level of 10 mg/kg. 
The donor organ is removed from the donor and perfused with a solution of 
electrolyte containing 0.5 mg/ml CAD for 10 minutes for 2 days before 
implantation into the recipient. 
In the post operative period, CAD treatment is continued at 10 mg/kg/day, 
I.V., to prevent the onset of rejection. The CAD is used in conjunction 
with other immunoregulatory strategies, such as immunosuppressive drugs 
like cyclosporine and the like. If organ rejection is suspected, or 
diagnosed, the amounts of CAD and other immunoregulatory drugs are 
increased until the rejection process is altered, or preferably, reversed. 
EXAMPLE 3 
The Experiments of Example 1 are repeated with the change that in place of 
SCA, and equivalent amount of acetylated CA (ACA) is employed. Similar 
results would be observed. 
EXAMPLE 4 
The Experiments of Example 1 are repeated with the change that in place of 
SCA, and equivalent amount of isolated dimeric native CA prepared by the 
method of Sophianopoulos, et al in PREP. BIOCHEM., 11 (4), 413-435. If 
this material is tested by isoelectric focusing it would be observed to be 
over 80% of the desired dimeric material. Similar in vivo test results 
would be observed.