Compounds for inhibiting immune response

Novel ruthenium complexes for use as immunosuppressive agents to prevent or significantly reduce graft rejection in organ and bone marrow transplantation are described. The ruthenium complexes can also be used as an immunosuppressant drug for T-lymphocyte mediated autoimmune diseases, such as diabetes, and may be useful in alleviating psoriasis and contact dermatitis.

BACKGROUND OF THE INVENTION 
Replacement of defective or severely injured tissues and organs has been a 
medical objective as long as medicine has been practiced. Grafts from an 
individual to himself almost invariably succeed, and are especially 
important in the treatment of burn patients. Likewise, grafts between two 
genetically identical individuals almost invariably succeed. However, 
grafts between two genetically dissimilar individuals would not succeed 
without immunosuppressive drug therapies. The major reason for their 
failure is a T cell mediated immune response to cell-surface antigens that 
distinguish donor from host. 
Immunosuppressive agents are also indicated in the treatment of autoimmune 
diseases such as rheumatoid arthritis or type I diabetes mellitus. One 
particular condition worth mentioning here is psoriasis. This disease is 
characterized by erythematous patches of skin accompanied by discomfort 
and itching. Hyperplasia of the epidermis involving proliferation of 
keratinocytes is also a hallmark feature of psoriasis. An inflammatory 
component is suggested by: (i) the finding of lymphocytic infiltration of 
epidermis, and (ii) the fact that immunosuppressive agents such as 
cyclosporin and corticosteroids have beneficial effect on the disease. 
A number of drugs are currently being used or investigated for their 
immunosuppressive properties. Among these drugs, the most commonly used 
immunosuppressant is cyclosporin A. However, usage of cyclosporin has 
numerous side effects such as nephrotoxicity, hepatotoxicity and other 
central nervous system disorders. Thus, there is presently a need to 
investigate new immunosuppressive agents that are less toxic but equally 
as effective as those currently available. 
SUMMARY OF THE INVENTION 
This invention relates to novel ruthenium complexes that are useful as 
immunosuppressive agents to prevent or significantly reduce graft 
rejection in organ and bone marrow transplantation. The ruthenium 
complexes can also be used as an immunosuppressant drug for T lymphocyte 
mediated autoimmune diseases, such as diabetes, rheumatoid arthritis, 
multiple sclerosis, lupus erythematosus and steroid resistant asthma. 
In another aspect, other diseases with suspected inflammatory components, 
such as psoriasis, contact dermatitis and hyperplasia of the epidermis, 
can be treated with a ruthenium complex of this invention to alleviate 
symptoms associated with these disease states. 
DETAILED DESCRIPTION OF THE INVENTION 
This invention is based upon the discovery that ruthenium complexes can 
inhibit antigen specific T lymphocyte proliferation in vitro. The data 
suggest that ruthenium complexes have potential use as immunosuppressants 
to reduce undesirable immune responses in humans. Ruthenium complexes can 
be used to facilitate organ transplantation, and to treat human autoimmune 
disorders where the specific activation of T cells is responsible for, or 
contributes to the pathology and progression of the diseases, such as 
diabetes, rheumatoid arthritis, multiple sclerosis, lupus erythematosus 
and steroid resistant asthma. 
This invention pertains to novel ruthenium complexes that have 
immunosuppressive properties of the general formula: 
EQU RuM.sub.m B.sub.b T.sub.t !Z.sub.n 
wherein Ru is ruthenium having an oxidation state of 2, 3 or 4; 
wherein M is a monodentate ligand that is a heterocyclic aromatic amine; 
wherein m is 0, 1, 2, 3, 4 or 6; 
wherein b is 0, 1, 2 or 3; 
wherein t is 0, 1 or 2; 
wherein B is a bidentate ligand that is a heterocyclic aromatic amine; 
wherein T is a tridentate ligand that is a heterocyclic aromatic amine; 
wherein Z is a counterion, for example a counterion selected from the group 
consisting of F.sup.-, Cl.sup.-, Br.sup.-, I.sup.-, NO.sub.3.sup.-, 
NH.sub.4 .sup.+, NR.sub.4.sup.1+, PF.sub.6.sup.-, SO.sub.4.sup.-2, R.sup.1 
ImH.sup.+, BPh.sub.4.sup.- and ClO.sub.4.sup.- ; wherein Im is imidazole 
wherein n is 0, 1, 2, 3 or 4; and 
wherein R.sup.1 is a linear or branched alkyl of 1-4 carbon atoms or aryl; 
provided that the ligands cannot be pyridine or pyrazine or derivatives of 
these. 
The coordination sphere of the metal center may contain all six ligands 
(referred to as monodentate) to be equivalent or a mixture of different 
ligands. The mixture of ligands can consist of different monodentate 
ligands; a mixture of bidentate/monodentate in a ratio of 1:4 or 2:2; 
three bidentate ligands; a mixture of bidentate/tridentate/monodentate in 
a ratio of 1:1:1; two tridentate ligands; or tridentate/monodentate in a 
1:3 ratio. 
For the purposes of this application, the terms "monodentate", "bidentate" 
and "tridentate" will have their generally accepted meaning in the art. 
That is, a monodentate ligand is defined as a chemical moiety or group 
which has one potential coordinating atom. More than one potential 
coordinating atom is termed a multidentate ligand where the number of 
potential coordinating atoms is indicated by the terms bidentate, 
tridentate, etc. 
The ruthenium metal can have different oxidation states, e.g., Ru(II), 
Ru(III) or Ru(IV). The complex will also contain a counterion of 
appropriate charge to render the overall charge of the complex neutral. 
Suitable counterions for cationic complexes, include but are not limited 
to, halide (F.sup.-, Cl.sup.-, Br.sup.- or I.sup.-), SO.sub.4.sup.-2, 
PF.sub.6.sup.-, BPh.sub.4.sup.-, ClO.sub.4.sup.- and NO.sub.3.sup.-. 
Examples of suitable counterions for anionic complexes include but are not 
limited to NH.sub.4.sup.+, NR.sub.4.sup.1+ and R.sup.1 ImH.sup.+ where 
R.sup.1 is a linear or branched alkyl of 1 to 4 carbons or aryl group and 
Im is imidazole. 
In one embodiment, the ruthenium complex can comprise six monodentate 
heterocyclic aromatic amine ligands. Examples of suitable heterocyclic 
aromatic amine ligands include but are not limited to imidazole, triazole, 
pyrazole, quinoline, pyridazine, pyrimidine, quinoxaline, quinazoline and 
isoquinazoline. Derivatives of these ligands can also be incorporated into 
the complex in various combinations with the non-substituted ligands. A 
derivative is a ligand in which one or more of the hydrogen atoms has been 
substituted with a moiety, such as C1-C5 alkyl, C2-C4 alkenyl, hydroxy, 
nitro, amino, carboxyl, ester, di-C1-C4 alkyl amine, phenyl, benzyl, 
imidazole and combinations of these. Preferred ligands are imidazole 
derivatives having the general formula: 
##STR1## 
where R.sup.2 and R.sup.3 are independently selected from the group 
consisting of aryl, heteroaryl, linear and branched alkyl (e.g., 1 to 8 
carbons), --C(O)H, --COOR.sup.1, --CONR.sup.1, --COOH, H, Cl, Br, I 
NO.sub.2 and methyl; wherein R.sup.1 is defined above. 
Examples of preferred ruthenium complexes having monodentate ligands are 
shown below. 
Ru(Im).sub.6 !Cl.sub.2 where Im=imidazole 
Ru (1-MeIm).sub.6 !Cl.sub.2 where 1-MeIm=1-methyl imidazole 
Ru(1-MeIm).sub.6 !(PF.sub.6).sub.3 
Ru(1-MeIm).sub.6 !Cl.sub.3 
Ru(Im).sub.6 !Cl.sub.3 
General procedures for making ruthenium complexes having six monodentate 
ligands are described in the exemplification section. 
In another embodiment, a ruthenium complex can be made having multidentate 
ligands, in combination with other multidentate ligands and/or monodentate 
ligands. Suitable heterocyclic aromatic amine bidentate ligands will 
include, but are not limited to, imidazole based ligands (e.g., 
2,2'-bis-(1-methylimidazolyl)phenylhydroxymethane); pyrazole based ligands 
(e.g., potassium-bis-pyrazolyl borate, bispyrazolyl methane); quinoline 
based ligands (e.g., 2,2'-bis(quinolinyl)phenylmethoxymethane); and 
quinazoline based ligands (2,2'-bis-(quinazolinyl)phenylmethoxymethane). 
Preferred are imidazole based ligands having the general formula: 
##STR2## 
where each R.sup.4 to R.sup.9 may be the same or different and are 
independently selected from the substituents defined above for R.sup.2 and 
R.sup.3. 
Examples of tridentate aromatic heterocyclic amine ligands include 
imidazole based ligands (e.g., bis-(2,-imidazolylmethyl)amine); pyrazole 
based ligands (e.g., potassium tris pyrazolyl borate); quinoline based 
ligands (e.g., 2,2'-bis-(quinolinylmethyl)amine, 
tris-(quinolinyl)methane). 
It has now been discovered that the ruthenium complexes of this invention 
possess immunosuppressive activity as confirmed through a drug screen. 
Specific T cell proliferation was measured in response to antigen exposure 
in the presence or absence of ruthenium complexes. It was found that 
ruthenium complexes inhibited T cell proliferation by 50% (IC.sub.50) at a 
concentration of about 10 to 100 nM. This compares favorably with 
cyclosporin A, which has an IC.sub.50 at 15 nM. In an in vitro toxicity 
study, ruthenium complexes were found to be nontoxic to a Jurkat cell line 
when tested at the same concentrations that markedly inhibit T cell 
activation (Table 1). Additional ruthenium complexes that have 
immunosuppressive capability are described in U.S. patent application 
entitled "Methods for Inhibiting Immune Response" U.S. Ser. No. 
08/331,204, filed Oct. 28, 1994, the entire teachings of which are 
incorporated herein by reference. 
Ruthenium complexes can be administered orally, parenterally (e.g. 
intramuscularly, intravenously, subcutaneously), topically, nasally or via 
slow releasing microcarriers in dosage formulations containing a 
physiologically acceptable vehicle and optional adjuvants and 
preservatives. Suitable physiologically acceptable vehicles include 
saline, sterile water, creams, ointments or solutions. 
Ruthenium complexes can be applied topically as a cream or ointment to 
locally deliver immunosuppressive concentrations of the drug without 
significant systemic exposure. Topical application may be the ideal way to 
deliver the compound in psoriasis and perhaps other inflammatory skin 
diseases such as contact dermatitis and pemphigus vulgaris. 
The specific dosage level of active ingredient will depend upon a number of 
factors, including biological activity of the ruthenium complexes, age, 
body weight, sex, general health, severity of the particular disease to be 
treated and the degree of immune suppression desired, as well as 
appropriate pharmacokinetic properties. It should be understood that 
ruthenium complexes can be administered to mammals other than humans for 
immunosuppression of mammalian autoimmune diseases. 
Ruthenium complexes can be administered in combination with other drugs to 
boost the immunosuppressive effect. Compounds that can be coadministered 
include steroids (e.g. methyl prednisolone acetate), NSAIDS and other 
known immunosuppressants such as azathioprine, 15-deoxyspergualin, 
cyclosporin, mizoribine, mycophenolate mofetil, brequinar sodium, 
leflunomide, FK-506, rapamycin and related molecules. Dosages of these 
drugs will also vary depending upon the condition and individual to be 
treated. 
The assay used to measure T cell growth inhibition was a human peripheral 
blood lymphocyte (PBL) proliferation assay using standard procedures known 
in the art. PBL's were chosen due to their known ability to proliferate in 
the presence of antigens derived from herpes simplex virus (HSV), Rubella 
or tetanus toxoid (TT). PBL growth inhibition was measured in terms of 
ruthenium complexes's ability to interfere with antigen induced lymphocyte 
proliferation. 
Ruthenium complexes can be used to produce antibodies (e.g., polyclonal and 
monoclonal) against the complexes. Methods for making antibodies are well 
known. The antibodies can be used as a diagnostic tool for monitoring the 
amount of ruthenium complex in patient blood levels. The ability to 
closely monitor the amount of ruthenium complex provides a suitable means 
for controlling drug delivery to patients in both preclinical and clinical 
settings. 
The invention will be further illustrated by the following non-limiting 
Examples:

EXAMPLE 1--Preparation of Ru(1-MeIm).sub.6 !Cl.sub.2 
RuCl.sub.3 (1.871 g, 9.04 mmol) was added slowly to 1-MeIm (10 mL, 125 
mmol, 14 eq.). The mixture was placed in a preheated oil bath (230.degree. 
C.), and the mixture was refluxed for 2 hours. The mixture was cooled down 
to room temperature and acetone (50-70 mL) was added to the mixture. The 
mixture was filtered and the solid washed with acetone (3.times.10 mL). 
The product was dried under vacuum. 
The product was dissolved in MeOH (30 mL), and filtered over celite. The 
product was obtained as a light yellow crystalline (3.27 g, 55%) solid 
after triple crystallization from MeOH/ether. 
Ru(1-MeIm).sub.6 !Cl.sub.2 was characterized by X-ray crystallography, 1H 
NMR, UV/Vis and elemental analysis. 
EXAMPLE 2--Preparation of Ru(1-MeIm).sub.6 !Cl.sub.3 
Ru(1-MeIm).sub.6 !Cl.sub.2 (0.405 g, 0.609 mmol) was dissolved in HCl 
(0.25M, 30 mL) and H.sub.2 O.sub.2 was added slowly until the starting 
material had disappeared (reaction followed by UV/Vis spectroscopy). The 
solvent was removed to dryness and the product was purified by 
recrystallization from MeOH/ether. The product was characterized by 
UV/Vis. 
EXAMPLE 3--PBL Antigen Specific Proliferation Assay 
The lymphocytes were prepared by first separating them from the blood 
samples of several donors by Ficoll gradient separation as described by 
standard procedure known in the art. The isolated lymphocytes were then 
grown in RPMI 1640 medium containing 5% human AB serum, glutamine (2 mM), 
penicillin/streptomycin, 50 .mu./ml/50 .mu.g/ml sodium pyruvate (1 mM) and 
HEPES buffer (10 mM). 
For assay purposes, PBL's were incubated at a density of 10.sup.5 per 200 
.mu.l of medium per well of a 96-well plate. 
Tetanus toxoid (TT; Connaught Labs, Willow Dale, ON) was used as a 
stimulating antigen at a concentration of 5 LF/ml. 
The test wells containing PBL's, were exposed to tetanus toxoid antigen, 
along with various dilutions of the ruthenium complexes solutions, as 
shown in Table 1. 
Subsequently, TT antigen/ruthenium complexes exposed PBL's were pulsed with 
1 .mu.Ci/well of .sup.3 H-thymidine on day 5 using a standard procedure 
known in the art. The cells were then harvested 16 hours later onto a 
glass fiber filter using a TOMTEC cell harvester. Thymidine incorporation 
was measured by liquid scintillation counting using a Beta plate counter 
(Pharmacia, Inc., Piscataway, N.J.). 
The results of the assay are shown in Table 1. 
TABLE 1 
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Cytotoxicity 
IC.sub.50 (Jurkat cell) 
Structure (.mu.g/mL IC.sub.50 (.mu.g/mL) 
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Ru(1-MeIm).sub.6 !Cl.sub.2 
0.052 .+-. 0.03 
2000 
Ru(1-MeIm).sub.6 !(PF.sub.6).sub.3 
0.19 .+-. 0.16 
115 
Ru(1-MeIm).sub.6 !Cl.sub.3 
0.12 .+-. 0.1 
&gt;300 
Ru(Im).sub.6 !Cl.sub.2 
0.0067 .+-. 0.003 
200 
Ru(4-MeIm).sub.6 !Cl.sub.2 
0.09 .+-. 0.07 
1040.sup.a 
Ru(Im).sub.6 !Cl.sub.3 
0.005 .+-. 0.004 
530.sup.a 
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a. values are extrapolated 
EQUIVALENTS 
Those skilled in the art will recognize, or be able to ascertain, using no 
more than routine experimentation many equivalents to the specific 
embodiments of the invention described herein. Such equivalents are 
intended to be encompassed by the following claims: