JP2002535358A - Methods for preventing graft rejection and ischemia-reperfusion injury - Google Patents

Abstract

  (57) [Summary] Methods for inhibiting rejection of transplanted grafts are disclosed. Administering to the transplant recipient an effective amount of a CCR1 function antagonist. Also disclosed is a method of inhibiting ischemia / reperfusion injury, comprising administering to a subject in need thereof an effective amount of a CCR1 function antagonist. The disclosed methods can further include co-administration of one or more adjuvant therapeutic agents, eg, an immunosuppressive agent and a cell adhesion inhibitor.

Description

DETAILED DESCRIPTION OF THE INVENTION

Related Application This application is a continuation-in-part of US patent application Ser. No. 09 / 239,283 (filed Jan. 29, 1999), the entire teachings of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION [0002] In many cases, the best and only treatment available for patients suffering from certain end-stage degenerative conditions or congenital genetic abnormalities is to transplant healthy grafts (eg, organs, tissues). is there. Advances in surgical techniques and post-operative immunosuppressive therapy have alleviated some of the obstacles to long-term survival of grafts and graft recipients, making this previously experimental treatment a more extensive clinical practice. Led to.

[0003] A major obstacle to the long-term survival of transplanted grafts is rejection by the recipient's immune system. Graft rejection is a hyperacute rejection mediated by preformed antibodies present in the recipient's circulatory system that can bind to the graft.
It can be classified as acute rejection mediated by the recipient's cellular immune response, or chronic rejection caused by a multifactorial process involving the immune component. The most notable major histocompatibility antigen (MHC), also known as tissue typing, as well as matching donor and recipient blood types, matches allelic variants of cellular antigens, resulting in hyperacute rejection The occurrence of the reaction is reduced. However, most transplants that are transplanted are not perfectly compatible with the recipient's histology (eg, allografts) and cannot survive without therapeutic intervention.

[0004] Allograft rejection is most notably due to drugs that inhibit calcineurin (eg, cyclosporin A (CsA), FK-506) for long-term (eg, life-long) prophylactic immunosuppression. It can be suppressed by therapy. Immunosuppressive therapy not only inhibits graft rejection, but also makes the recipient susceptible to, for example, viruses, bacteria and fungi (eg, yeast, mold) and is at risk for the development of some malignancies To a state where has become larger. In addition, therapeutic doses of immunosuppressive drugs can cause adverse side effects such as diabetes mellitus, neurotoxicity, nephrotoxicity, hyperlipidemia, hypertension, hirsutism and gingival hyperplasia (Spencer, C.
. M. Et al., Drugs, 54 (6): 925-975 (1997)). Therefore,
The degree of immunosuppression must be carefully adjusted to prevent transplant rejection and maintain the overall health of the recipient.

[0005] Despite such prophylactic immunosuppression, acute and chronic rejection of transplants remains a clinical problem. Acute manifestations of rejection are characterized by graft infiltration by recipient leukocytes (eg, monocytes, macrophages, T cells), and cell necrosis. These episodes usually occur between days and months after transplantation. Acute rejection can be caused by certain immunosuppressants such as glucocorticoids (eg, prednisone) and certain antibodies that bind to leukocytes (eg, OKT3).
) At high doses. However, this treatment does not always stop rejection, is associated with systemic side effects, and may lose efficacy if the rejection recurs.

[0006] Chronic rejection is a major cause of transplant failure and recipient death for those patients who survive one year. Evidence of chronic rejection can be found in about 40% to 50% of heart and / or lung allograft recipients who have survived for 5 years, with most kidney transplants not tolerating chronic rejection. The etiology of chronic rejection is complex, with accelerated atherosclerosis of the vasculature and leukocyte infiltration associated with the graft (
For example, atherosclerosis) is involved. Unlike acute rejection episodes, chronic rejection generally does not respond to additional immunosuppressive treatments. Furthermore, the accelerated atherosclerosis of grafts characteristic of chronic rejection is generally sporadic, making conventional treatments (eg, angioplasty, bypass grafts, endarterectomy) easier. Not accepted. Therefore, patients who chronically reject those transplants require a second day transplant (Schroeder, JS "Heart Transplant", 1
298-1300; Maurer, J. et al. R. "Lung Transplant", pp. 1491-14
P. 93; Carpenter, C .; B. And Lazarus, J .; M. "Dialysis and Transplantation in the Treatment of Renal Failure", pp. 1524-1529; Dienstag,
J. "Liver transplantation", pp. 1721-1725; all of which are Harriso
n's Principles of Internal Medicine,
14th Edition, ed., Edited by Fauci et al., McGraw Hill (1998)).

[0007] Oxygenated blood flow to all implanted grafts is stopped during graft procurement and transplant surgery. The cessation of blood results in hypoxia and necrosis of some or all of the cells of the graft, depending on how long the blood flow has stopped. The resulting damage or injury appears after the blood flow returns (reperfusion). This type of injury, called ischemia / reperfusion injury, can result in the death of endothelial cells lining the blood vessels (eg, arteries) associated with the graft (Gohra, H. et al., Transplan
Tation, 60 (1): 96-102 (1995)). Thus, ischemia / reperfusion injury can be a contributing factor to accelerated arteriosclerosis of the graft and graft rejection.

[0008] There is a need for treatments to prevent graft rejection and ischemia / reperfusion injury.

SUMMARY OF THE INVENTION [0009] The present invention relates to transplantation and to enhancing the survival of the implanted graft.
In one concept, the invention relates to a method for inhibiting (reducing or preventing) transplant rejection (eg, acute rejection, chronic rejection). In one aspect, the method comprises administering to the transplant recipient an effective amount of a CCR1 function antagonist. In other embodiments, the implant is an allograft.
In certain embodiments, the allograft is a heart. In a preferred embodiment, the method comprises administering to the transplant recipient a CCR1 function antagonist and one or more immunosuppressive agents.

In another aspect, the invention is directed to a method for inhibiting (reducing or preventing) ischemia / reperfusion injury. In one aspect, the method comprises administering to a subject in need thereof an effective amount of a CCR1 function antagonist. In some embodiments, ischemia / reperfusion injury is the result of trauma or a medical procedure, eg,
It can be the result of surgery. In other embodiments, the ischemia / reperfusion injury can be the result of a pathological condition, for example, the result of atherosclerosis, myocardial infarction, stroke, or a transient ischemic stroke. In certain embodiments, the ischemia / reperfusion injury is the result of a graft transplant. In another particular embodiment, the implant is a kidney. In another embodiment, the method comprises providing a graft recipient with a CCR1 function antagonist,
More than one immunosuppressant (eg, thrombolytics, cell adhesion inhibitors, anticoagulants,
An antithrombotic agent and an activator or inhibitor of nitric oxide synthase).

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to transplantation and the viability of the transplanted graft. In particular, the present invention provides for transplant rejection (eg, acute graft rejection) by administering to a graft recipient an effective amount of an antagonist of a mammalian (eg, human) CC chemokine receptor CCR1. , Chronic graft rejection).

Chemokines are a family of pro-inflammatory mediators that promote the recruitment and activation of multilineage leukocytes (eg, lymphocytes, macrophages). Chemokines can be released by many types of tissue cells after activation. Continuous release of chemokines at sites of inflammation may mediate continued migration and recruitment of effector cells to sites of chronic inflammation. Chemokines are related in primary structure and have four conserved cysteines that form disulfide bonds.
Based on this conserved cysteine motif, the families can be divided into different groups, including CXC chemokines (α-chemokines) and CC chemokines (β-chemokines), each of which is , The first two conserved cysteines are separated by, or adjacent to, intervening residues (Ba
ggiolini, M .; And Dahinden, C .; A. , Immunolog
y Today, 15: 127-133 (1994)).

[0013] CXC chemokines include many potent chemoattractants of neutrophils, such as interleukin 8 (IL-8), PF4 and neutrophil activating peptide-2 (NAP-2). And activators. As CC chemokines, for example, R
ANTES (R egulated on A ctivation, N ormal T E xpressed and S ecreted), the macrophage inflammatory proteins l [alpha] and 1β (MIP-1α and MIP-1β), Eotakushin and human monocyte chemotactic proteins 1 to 3 (MCP 1, MCP-2, MCP-3), which have been characterized as chemoattractants and activators of monocytes or lymphocytes. Chemokines such as RANTES and MIP-1α have been implicated in acute and chronic inflammatory diseases in humans, including respiratory diseases such as asthma and allergic diseases.

[0014] Chemokine receptors are members of the G protein-coupled receptor (GPCR) superfamily with structural features that reflect a common mechanism of signal transduction (Gerard, C. and Gerard, NP, Annu.Rev.I
mmunol. Gerard, C., 12: 775-808 (1994); And Gerard, N.M. P. Curr. Opin. Immunol. 6: 140-
145 (1994)). Conserved features include seven hydrophobic domains that span the plasma membrane and are connected by hydrophilic extracellular and intracellular loops. Large primary structural homology is present in the hydrophobic transmembrane region, and the hydrophilic regions are more different. Receptors for CC chemokines include, for example, MIP
-1α, RANTES, MCP-2, MCP-3, MCP-4, CKbeta8
, CKbeta8-1, Leukotactin-1, HCC-1 and CCR1 capable of binding to MPIF-1; for example, MCP-1, MCP-2, MCP-3 and MCP-
CCR2 capable of binding to C.4; e.g. eotaxin, eotaxin-2, RANT
CCR3 capable of binding to ES, MCP-2, MCP-3 and MCP-4;
CCR4 capable of binding to TARC, RANTES, MIP-1α and MCP-1;
For example, CCR5 capable of binding to MIP-1α, RANTES and MIP-1β;
For example, CCR6 capable of binding LARC / MIP-3α / Exodus;
CCR7 capable of binding to ELC / MIP-3β; and CCR8 capable of binding to I-309 (Baggiolini, M., Nature, 3).
92: 565-568 (1998); Luster, A .; D. , New Eng
land Journal of Medicine, 338 (7): 436-
445 (1998); Tsou et al. Exp. Med. , 188: 603-6.
08 (1998); Nardelli et al., J Immunol. , 162 (1)
: 435-444 (1999); Youn et al., Blood, 91 (9): 311.
8-3126 (1998); Youn et al., J Immunol. , 159 (11
): 5201-5201 (1997)).

[0015] CCR1 and the processes and cellular responses mediated by CCR1 are involved in the rejection of transplanted grafts. As described herein,
Allograft survival studies were performed using a murine heart transplant model. CCR
Mice lacking the functional chemokine receptor CCR1 as a result of targeted disruption of one gene (CCR1 KO; Gerard, C. et al., J. Clin. Inves
t. , 100: 2022-2027 (1997)) describe MHC class I and MH
Rejection to transplanted grafts that did not match C class II was as fast as control mice that contained a functional CCR1 gene and were otherwise genetically identical to CCR1 KO mice. Did not occur (Example 1, Table 1, Group 1
And group 2).

As described herein, by giving low doses of immunosuppressant therapy (cyclosporin A) to CCR1 + / + control mice, untreated CCR1 + /
+ Compared with animals, the survival of the graft was only increased by 2 to 3 days (Example 1, Table 1, Group 3). Surprisingly, by administering the same low dose of CsA with inhibition of CCR1 function, permanent engraftment (> 100 days) can be up to 2 days.
Obtained in CCR1 KO mice given CsA for only one day (Table 1,
Group 4).

[0017] In a further study, allografts incompatible with only MHC class II were transplanted into CCR1 KO mice and CCR1 + / + control mice. As expected, partial histocompatibility resulted in longer survival of the graft in CCR1 + / + control mice. However, all CCR1 + / + control mice
By day 35, rejection of the grafts was again noted (Example 1, Table 1, group 5).
). By partially adapting the MHC antigen with inhibition of CCR1 function,
Permanent (> 100 days) engraftment was obtained as well as low dose immunosuppression (Example 1, Table 1, Group 6).

Histological examination (see Table 1) of permanently engrafted hearts isolated from group 4 and group 6 mice 100 days after transplantation revealed only minimal infiltration of mononuclear cells. ,
No evidence of accelerated arteriosclerosis of the implant was revealed.

[0019] The survival of class I and class II incompatible allografts can be prolonged by administering anti-CD4 monoclonal antibodies (mAbs) (Mott).
ram et al., Transplantation, 59: 559-565 (1995).
)). However, the long-term survival of these grafts is complicated by the development of chronic rejection with extensive arteriosclerosis in the graft vasculature (Ha
ncock et al., Nature Medicine, 4: 1392-1396 (1
998)).

As described herein, class I and class II incompatible allografts were obtained from anti-CD4 mAb-treated CCR1 KO mice and CCR1 + /
+ Survived in control mice for 60 days. Morphological examination of grafts removed from CCR1 + / + control recipients at day 60 revealed severe arteriosclerosis. In contrast, grafts taken from CCR1 KO recipients did not show evidence of arteriosclerosis (Example 2, Table 2). Thus, disruption of CCR1 function may provide a dual effect of inhibiting both acute and chronic rejection of allografts.

Accordingly, a first aspect of the present invention provides a transplant recipient with an effective amount of a CCR.
Provided are methods of inhibiting graft rejection (eg, acute and chronic rejection) comprising administering a monofunctional antagonist.

CCR1 Antagonist As used herein, the term “CCR1 functional antagonist” refers to an agent (eg, molecule, compound) that can inhibit the function (ie, one or more) of CCR1.
For example, a CCR1 functional antagonist may include one or more ligands (eg, MIP
-1α, RANTES, MCP-2, MCP-3, MCP-4, CKbeta8
, CKbeta8-1, leukotactin-1, HCC-1, MPIF-1)
It can inhibit binding to CR1 and / or inhibit signaling mediated through CCR1 (eg, GDP / GTP exchange by CCR1-related G proteins, intracellular calcium flux). Thus, processes and cellular responses mediated by CCR1 (eg, proliferation, migration, chemotactic response, secretion or degranulation)
Can be inhibited by CCR1 function antagonists.

Preferably, the CCR1 functional antagonist is a compound that is, for example, a small organic molecule, natural product, protein (eg, antibody, chemokine, cytokine), peptide or peptidomimetic. Several molecules are known in the art that can neutralize one or more functions of a chemokine receptor (eg, CCR1). This includes, for example, International Patent Application Publication WO97 / 24325; Pf by Takeda Pharmaceutical Company Limited.
iser, Inc. WO98 / 38167; WO97 / by Teijin Limited
44329; WO98 / 04554 by Banyu Pharmaceutical Co., Ltd .; Merck & Co,
, Inc. WO98 / 27815, WO98 / 25604, WO98 / 2
5605, WO98 / 25617 and WO98 / 31364; LeukoSit
e, Inc. WO98 / 02151 and WO99 / 37617; Leu
koSite, Inc. WO99 / 37651 and WO99 / 3761.
9; U.S. Provisional Application No. 60 / 021,716 (filed on July 12, 1996); U.S. Patent Application No. 09 / 146,827 and 09 / 148,2.
No. 36 (filed on Sep. 4, 1998); Hesselgesser et al.
Biol. Chem. 273 (25): 15687-15692 (1998);
And Howard et al. Medicinal Chem. 41 (13): 21
84-2193 (1998); antibodies (eg, polyclonal serum, monoclonal, chimeric, humanized) and antigen-binding fragments thereof (eg, Fab, Fab ', F (ab') ₂ , Fv). ), For example, S
u et al. Leukocyte Biol. 60: 658-656 (1996)
Variants and analogs of chemokines, for example, US Pat. No. 5,739,103 (provided to Rollins et al.), WO 96/385.
59 (Dana Farber Cancer Institute) and WO
98/066751 (Research Corporation Techno
logs, Inc. ); Peptides such as WO98 / 0
9642 (United States of America). The entire teachings of each of the patent applications and references cited above are incorporated herein by reference.

A CCR1 functional antagonist may be screened from a molecular library or collection, such as, for example, a National Cancer Institute (United States) chemical repository, as described herein, or otherwise. It can be identified by using a suitable method.

Another source of CCR1 function antagonists is combinatorial libraries, which can contain many structurally distinct molecular species. Combinatorial libraries can be used to identify lead compounds or to optimize previously identified lead compounds. Such libraries can be produced by well known methods of combinatorial chemistry, and can be screened by any suitable method, such as the methods described herein.

The term “natural product” as used herein is found in compounds found in nature, for example, naturally occurring metabolites of marine organisms (eg, capsids, algae) and plants. The resulting compound refers to a compound having biological activity, for example, a compound capable of neutralizing CCR1 function. For example, lactacystin, paclitaxel and cyclosporin A are natural products that can be used as antiproliferative or immunosuppressive agents.

[0027] Natural products can be isolated and identified by any suitable means. For example, a suitable biological source (eg, a plant) can be homogenized (eg, by milling) in a suitable buffer and clarified by centrifugation, thereby producing an extract. Can be. The resulting extract can be assayed for its ability to neutralize CCR1 function, for example, by the assays described herein. C
Extracts containing activities that neutralize CR1 function can be purified by any suitable method, such as fractionation (eg, column chromatography (eg, ion exchange, reverse phase, affinity), phase partitioning, fractional crystallization), and biological techniques. Can be further processed to isolate CCR1 antagonists by assaying for specific activity (eg, neutralizing effects of CCR1 activity). Once isolated, the structure of the natural product can be determined (eg, by nuclear magnetic resonance (NMR)), and one of ordinary skill in the art can devise a synthetic scheme for synthesizing the natural product. Thus, natural products can be isolated from nature (
For example, it can be substantially purified), or it can be fully or partially synthesized. Natural products can be modified (eg, derivatized) to optimize their therapeutic potential. Thus, the term "natural product" as used herein includes those compounds obtained using standard medicinal chemistry techniques to optimize the therapeutic potential of a compound that can be isolated from nature. Compounds.

As used herein, the term “peptide” refers to a compound consisting of about 2 to about 90 amino acid residues in which the amino group of an amino acid is linked to the carboxyl group of another amino acid by a peptide bond. Peptides can be obtained from, or removed from, native proteins, for example, by enzymatic or chemical cleavage, or can be obtained using conventional peptide synthesis techniques (eg, solid phase synthesis) or molecular biology techniques (Sa
mbrook, J .; Et al., Molecular Cloning: A Labor
attory Manual, Cold Spring Harbor Pres
s, Cold Spring Harbor, NY (1989)). "Peptide" refers to any suitable L-amino acid and / or
Or D-amino acids, such as common α-amino acids (eg, alanine, glycine, valine), non-α-amino acids (eg, β-alanine, 4-aminobutyric acid, 6-
Aminocaproic acid, sarcosine, statins) and unusual amino acids (unusual amino)
acid) (eg, citrulline, homocitrulline, homoserine, norleucine, norvaline, ornithine). The amino, carboxyl, and / or other functional groups of the peptide may be free (eg, unmodified) or protected with a suitable protecting group. Suitable protecting groups for amino and carboxyl groups and means for adding or removing protecting groups are known in the art, for example,
Green and Wuts, "Protecting Groups in Or.
Gannic Synthesis ", John Wiley and Sons
, 1991. Peptide functional groups can also be derivatized (eg, alkylated) by methods known in the art.

Peptides can be synthesized and assembled into a library containing a small number to a large number of different molecular species. Such libraries can be prepared using well known methods of combinatorial chemistry and screened for CCR1 function as described herein or using other suitable methods. It can be determined whether a compatible peptide is included in the library. Such peptide antagonists can then be isolated by any suitable method.

The term “peptidomimetic” as used herein refers to molecules that are not polypeptides, but mimic their structural aspects. For example, a polysaccharide having the same functional group as a peptide capable of neutralizing CCR1 can be synthesized. Peptide mimetics can be designed, for example, by binding to CCR1 or establishing the three-dimensional structure of a peptide agent in an environment that binds CCR1. The peptidomimetic is
It comprises at least two components: one or more binding moieties and a skeletal or support structure.

[0031] The binding moieties are chemical atoms that form a reaction or complex with CCR1, eg, with or near amino acids at the ligand binding site (eg, by hydrophobic or ionic interactions). Or a chemical group. For example, the binding moiety in the peptidomimetic can be the same as the binding moiety in the peptide antagonist of CCR1. The binding moiety is CCR1
May be atoms or chemical groups that react with the receptor in the same or similar manner as the binding moieties in the peptide antagonists. Examples of suitable linking moieties used in designing peptidomimetics for basic amino acids in the peptide are nitrogen-containing groups such as amines, ammonium, guanidine and amide or phosphonium. Examples of suitable linking moieties used in designing peptidomimetics for acidic amino acids include, for example, carboxyl, lower alkyl carboxylate, sulfonic acid,
It may be a lower alkyl sulfonic acid ester or phosphorous acid or an ester thereof.

A support structure is a chemical moiety that, when attached to one or more binding moieties, results in a three-dimensional configuration of the peptidomimetic. The support structure can be organic or inorganic.
Examples of organic support structures include polysaccharides, polymers or oligomers of organic synthetic polymers (such as polyvinyl alcohol or polylactide), and support structures that are substantially the same size and bulk as the peptide backbone structure or support structure.
ons). This can be determined by calculating or measuring the size of the atoms and bonds of the peptides and peptidomimetics. In one embodiment, the nitrogen of the peptide bond can be replaced with oxygen or sulfur,
Thereby, a polyester skeleton can be formed. In another aspect, the carbonyl can be replaced with a sulfonyl or sulfinyl group, thereby forming a polyamide (eg, a polysulfonamide). Reverse amide of the peptide (eg, one or more -CON
(H-group replaced by -NHCO- group). In yet another aspect, the peptide backbone can be replaced with a polysilane backbone.

[0033] These compounds can be produced by a known method. For example, a polyester peptide mimetic may optionally block basic and acidic side chains to minimize side reactions, replacing the corresponding α-amino group in an amino acid with a hydroxyl group, To prepare a hydroxy acid, followed by esterification of the hydroxy acid. Determining the appropriate chemical synthesis route generally involves determining
It can be easily recognized when the chemical structure is determined.

[0034] Peptide mimetics can be synthesized and assembled into libraries containing a small number to a large number of different molecular species. Such libraries can be prepared using well-known methods of combinatorial chemistry, and screened as described herein to obtain one or more peptidomimetics that neutralize CCR1 function. Can be determined. The peptidomimetic antagonist can then be isolated by any suitable method.

In one embodiment, the CCR1 antagonist is an antibody having specificity for CCR1 or an antigen-binding fragment thereof. Antibodies can be polyclonal or monoclonal. The term "antibody" is intended to include both polyclonal and monoclonal antibodies. The terms polyclonal and monoclonal are
Indicates the homogeneity of the antibody preparation and is not limited to a particular production method. Also,
The term “antibody” as used herein encompasses functional fragments of the antibody. This includes chimeric antibodies, humanized antibodies, primatized antibodies, veneered antibodies or fragments of single chain antibodies. The functional fragment is C
Includes antigen-binding fragments that bind to CR1. For example, but not limited to, Fv, Fa
Antibody fragments which can bind to CCR1 or portions thereof, including fragments of b, Fab 'and F (ab') ₂ , are encompassed by the present invention. Such fragments can be made by enzymatic cleavage or by recombinant techniques. For example, cleavage with papain or pepsin can yield Fab or F (ab ') ₂ fragments, respectively. Alternatively, Fab fragments or F (ab ') ₂ fragments can be obtained using other proteases having the required substrate specificity. Antibodies can also be produced in various truncated forms using an antibody gene in which one or more stop codons have been introduced upstream of the natural stop site. For example, a chimeric gene encoding the heavy chain portion of the F (ab ') _2, C
It can be designed to include DNA sequences encoding the H1 domain and the hinge region of the heavy chain. Single chain antibodies, as well as chimeric, humanized or primatized (CDR-grafted) or veneered antibodies containing portions from different species,
And chimeric single-chain antibodies, CDR-grafted single-chain antibodies or veneered single-chain antibodies and the like are also encompassed by the present invention and the term “antibody”. The various portions of these antibodies can be chemically linked by conventional techniques, or can be prepared as a continuous protein using genetic engineering techniques. For example, a nucleic acid encoding a chimeric or humanized chain can be expressed to produce a continuous protein. For example, Cabilly et al., US Pat. No. 4,816,567; Cabilly et al., EP 0,125,023B1; Bo.
No. 4,816,397; Boss et al., European Patent No.
120, 694B1; Neuberger, M .; S. Et al., WO86 / 0
1533; Neuberger, M .; S. Et al., European Patent 0,194,276B.
No. 1, Winter; U.S. Pat. No. 5,225,539; Wint
er, EP 0,239,400 B1; Queen et al., EP 0 451 216 B1; and Padlan, E. et al. A. Et al. EP05
See 19 596 A1. For primatized antibodies, see Newma
n, R. Et al., BioTechnology, 10: 1455-1460 (199).
2), for single chain antibodies, Ladner et al., US Pat. No. 4,946,778.
And Bird, R.A. E. FIG. Et al., Science, 242: 423-426.
(1988).

[0036] Humanized antibodies can be prepared using synthetic techniques or recombinant DNA techniques using standard or other suitable techniques. Also, a nucleic acid (eg, cDNA) sequence encoding a humanized variable region may be a DNA sequence encoding a human or humanized chain, such as a DNA template obtained from a previously humanized variable region.
Can be constructed using PCR mutagenesis to alter the NA sequence (eg,
Kamman, M .; Et al., Nucl. Acids Res. , 17: 5404 (1
Sato, K. 989); Et al., Cancer Research, 53: 851.
-856 (1993); Daugherty, B .; L. Et al., Nucleic A
cids Res. , 19 (9): 2471- 2476 (1991); Lewi.
s, A. P. And J.A. S. Crowe, Gene, 101: 297-302 (1
991)). Also, using these methods or other suitable methods,
Variants can be easily produced. In one embodiment, the cloned variable region can be mutated and a sequence encoding a variant with the desired specificity [eg, from a phage library (eg, Krebbe).
et al., U.S. Patent No. 5,514,548; Hoogenboom et al., WO
93/06213, published Apr. 1, 1993)].

Antibodies specific for mammalian (eg, human) CCR1 can be isolated from a suitable immunogen, such as isolated and / or recombinant human CCR1 or portions thereof (including synthetic molecules such as synthetic peptides). Can be raised. Antibodies can also be raised by immunizing a suitable host (eg, a mouse) with cells expressing CCR1, such as activated T cells (eg, the teachings of which are incorporated herein by reference). U.S. Pat. No. 5,440,020). further,
Cells expressing recombinant CCR1 such as transfected cells are used as immunogens.
Alternatively, they can be used in screens for antibodies that bind to the receptor (eg, Chuntarapai et al., J. Immunol., 152).
: 1783-1789 (1994); see Chuntarapai et al., U.S. Patent No. 5,440,021).

Preparation of the immunizing antigen and production of polyclonal and monoclonal antibodies can be performed using any suitable technique. Various methods have been described (e.g., Kohler et al., Nature, 256: 495-497 (1975).
) And Eur. J. Immunol. , 6: 511-519 (1976); Mi.
Istein et al., Nature, 266: 550-552 (1977); Kop.
Rowski et al., U.S. Patent No. 4,172,124; Harlow, E .;
And D. Lane, 1988, Antibodies: A Laboratory
y Manual (Cold Spring Harbor Laboratory)
ry: Cold Spring Harbor, NY); Current Pr
otocols In Molecular Biology, Volume 2 (Supplement 2
7, Summer 1994), Ausubel, F. et al. M. Hen (John Wiley &
Sons: New York, NY), Chapter 11 (1991)).
Generally, the hybridoma is a suitable immortal cell line (eg, SP2 / 0 or P3
X63Ag8.653) and antibody-producing cells. Antibody-producing cells, preferably antibody-producing cells obtained from the spleen or lymph nodes, can be obtained from animals immunized with the antigen of interest. Fusion cells (hybridomas) can be isolated using selective culture conditions and cloned by limiting dilution. Cells that produce antibodies with the desired specificity can be selected by a suitable assay (eg, ELISA).

Other suitable methods for producing or isolating antibodies of the required specificity, for example, selecting recombinant antibodies from a library (eg, a phage display library), or producing a human antibody repertoire A method by immunizing a transgenic animal (eg, mouse) to be obtained is used (eg, Jako).
bovits et al., Proc. Natl. Acad. Sci. USA, 90:25
51-2555 (1993); Jakobovits et al., Nature, 362.
: 255-258 (1993); Lonberg et al., U.S. Patent No. 5,545,8.
06; Surani et al., US Pat. No. 5,545,807; Lo.
nberg et al., WO 97/13852).

[0040] In one embodiment, the antibody or antigen-binding fragment thereof has specificity for a mammalian CC chemokine receptor-1 (CCR1), such as human CCR1. In a preferred embodiment, the antibody, or antigen-binding fragment thereof, binds the ligand (ie, one or more ligands) to CCR1 and / or responds to CC binding in response to ligand binding.
One or more functions mediated by R1 can be inhibited. Preferred antibody antagonists of CCR1 function are Shixin Qin, Walter New
"Anti-CCR1 antibody and its use" by man and Nasim Kassam
And co-pending U.S. Patent Application entitled "Attorney Docket No. LKS97."
-13, U.S. patent application Ser. No. 09 / 239,938, filed on Jan. 29, 1999)
And in International Patent Application No. PCT / US99 / 04527,
The entire teachings of each of these specifications are incorporated herein by reference.

Assessing Activity of Antagonists The ability of agents (eg, proteins, peptides, natural products, small organic molecules, peptidomimetics) to neutralize CCR1 function can be determined using suitable screening (eg, high-throughput assays). Can be determined. For example, the agent can be tested in an extracellular acidification assay, calcium flux assay, ligand binding assay or chemotaxis assay (eg, Hasselgesser et al., J. Bi).
ol. Chem. 273 (25): 15687-15692 (1998) and WO 98/02151).

In a particular assay, the membrane was coated on THP-1 cells (American Type).
(Culture Collection, Manassas, VA; Accession No. TIB202) and the like. The cells can be collected by centrifugation, washed twice with PBS (phosphate buffered saline), and the resulting cell pellet can be frozen at -70C to -85C. The frozen pellet was mixed with 5 mM HEPES (N-2-hydroxyethylpiperazine-N
'-2-ethanesulfonic acid, pH 7.5), 2 mM EDTA (ethylenediaminetetraacetic acid), 5 μg / ml each of aprotinin, leupeptin and chymostatin (
Thaw at a concentration of 1-5 × 10 ⁷ cells / ml in ice-cold lysis buffer consisting of protease inhibitor) and 100 μg / ml PMSF (phenylmethanesulfonyl fluoride—also a protease inhibitor). Can be achieved. The resulting suspension can be mixed well to resuspend all frozen cell pellets. Nuclear and cell debris can be removed by centrifugation at 400 × g for 10 minutes at 4 ° C. The resulting supernatant can be transferred to a new tube and the membrane fragments can be collected by centrifugation at 25,000 xg for 30 minutes at 4 ° C. The resulting supernatant is aspirated and the pellet is washed with 10 mM HEP.
ES (pH 7.5), 300 mM sucrose, 1 μg / ml each of aprotinin, leupeptin and chymostatin and 10 μg / ml PMSF can be resuspended in a freezing buffer (approximately 10 ⁸ cells per 10 ⁸ cells). 0.1 ml). All clumps can be redissolved using a mini-homogenizer and the total protein concentration determined by a suitable method (eg, Bradford assay, Lowery assay). Divide the membrane solution into small portions and until necessary -70 ° C-
It can be frozen at 85 ° C.

The membrane preparation described above can be used in a suitable binding assay.
Wear. For example, membrane protein (2-20 μg total membrane protein) is
nM to 0.2 nM¹²⁵Unlabeled competition with I-labeled RANTES or MIP-1α
Agent (RANTES or MIP-1α) or in the presence of various concentrations of the test compound or
Incubation can be in the absence.¹²⁵I-labeled RANTES and ¹²⁵ I-labeled MIP-1α can be prepared by any suitable method or can be obtained from commercial sources (eg, D
uPont-NEN (Boston, Mass.). The binding reaction
10 mM HEPES (pH 7.2), 1 mM CaCl_Two5 mM MgCl _Two And binding buffer consisting of 0.5% BSA (bovine serum albumin)
It can be performed at room temperature for 60 minutes in 00 μl. The binding reaction is 0.3% po
Glass fiber filters (eg GF) that can be presoaked in ethyleneimine
/ B or GF / C, by collecting the membrane by rapid filtration through Packard).
Can be stopped. Filter was added to approximately 600 μl containing 0.5 M NaCl.
Wash with binding buffer, dry and scintillation count the amount of bound radioactivity
Can be measured.

The CCR1 antagonist activity of a test agent (eg, a compound) is determined by the specific binding in a receptor binding assay (eg, using ¹²⁵ I-RANTES or ¹²⁵ MIP-1α as a ligand and THP-1 cell membrane). 50% inhibition (IC ₅₀ value)
As the inhibitor concentration required. Specific binding is preferably defined as total binding (eg, total cpm on the filter) minus non-specific binding. Non-specific binding is due to excess unlabeled competitor (eg, RANTES or MIP-1α).
) Is defined as the amount of cpm still detected in the presence of

If desired, membranes prepared from cells expressing recombinant CCR1 can be used in the assays described.

In addition, the ability of a compound to neutralize CCR1 function may be
It can be determined in a chemotaxis assay. Suitable cells include, for example, C
It expresses CR1 and is induced by a CCR1 ligand (eg, MIP-1
α, RANTES, MCP-2, MCP-3, MCP-4, HCC-1 or MP
IF-1) chemotactic cell lines, recombinant cells or isolated cells. One case
As an example, recombinant L1.2 cells expressing CCR1 (Campbell et al., JC
ell Biol, 134: 255-266 (1996)), butyric acid
Of Peripheral Blood Mononuclear Cells or HL60 Cells Differentiating by Transendothelial Cell Migration Assay
(Carr, MW, et al., TA, Pro.
c. Natl. Acad. Sci. USA, (91): 3652 (1994)).
. Peripheral blood mononuclear cells can be isolated by any suitable method, e.g., density gradient centrifugation, and specificity.
Whole blood by positive or preferably negative selection using
Can be isolated from The endothelial cells used in this assay are preferably Eu.
Ropean Collection of Animal Cell Cul
tures (Porton Down, Salisbury, UK)
Endothelial cell line ECV304. Endothelial cells have a pore size of 3.0 μm
A 6.5 mm diameter Transwell culture insert (Coster
Corp .. , Cambridge, MA). Of ECV304 cells
The culture medium may consist of M199 + 10% FCS, L-glutamine and antibiotics
You. Assay medium contains equal volumes of RPMI 1640 and M19 with 0.5% BSA.
May consist of nine. 2 × 10 2 hours before assay^Five24 ECV304 cells
Place in each insert of the well Transwell chemotaxis plate and incubate at 37 ° C.
Can be cubified. RANTES or MIP-1α (Pepro
chemotactic factors (diluted in assay medium) such as
Can be added to the plate in a final volume of 600 μL. Coated with endothelial cells
Transwells can be placed in each well and studied. ⁶ The upper chamber in a final volume of 100 μL of the assay medium
Is added to The plate was then placed at 37 ° C. with 5% CO 2_Two/ 95% air
Incubation can be for 1-2 hours. During incubation
Cells migrating to the head chamber are counted using, for example, flow cytometry
can do. Bottom to count cells by flow cytometry
500 μL of the cell suspension from the chamber can be placed in a tube and the relative
A typical count value can be obtained in a set time, for example, in 30 seconds. This meter
The method is highly reproducible, allows gating on leukocytes, and
From crushed or other cell types. On the other hand, the cells are
Can be counted. Assays for assessing chemotaxis inhibitors include adding cells
Before dissolving the antagonist in the assay medium containing up to 1% DMSO co-solvent
Except that liquid can be added to both upper and lower chambers
And can be performed in the same manner as the control experiment described above. Antagonis
The ability to migrate to the lower chamber in the wells containing the antagonist
Compare the number of cells to the number of cells migrating to the lower chamber in control wells.
And can be determined by: Control wells may contain equal amounts of DMSO, but
Does not include antagonists.

[0047] The activity of a CCR1 functional antagonist can also be assessed by monitoring the cellular response induced by the active receptor using suitable cells that express the receptor. For example, exocytosis (eg, esterase (
For example, 1 such as serine esterase), perforin and / or granzyme
Degranulation) of cells that results in the release of seeds or more enzymes or other granule contents, inflammatory mediator release (leukotrienes (e.g., leukotriene C ₄₎ such bioactive lipids, such as release)) and respiratory burst, art It can be monitored by methods known in the art or by other suitable methods (eg, for assays for release of serine esterase from granules, see Taub, DD, et al., J. Immunol.
. 155: 3877-3888 (1995); For assays on enzyme and granzyme release, see Loetscher et al. Immunol. , 1
56: 322-327 (1996); see Ret. J. et al. Exp. Med. 176: 1489-1495 (1992); Bisc.
Hoff, S.M. C. Et al., Eur. J. Immunol. , 23: 761-767.
(1993), and Baggliolini, M .; And C.I. A. Dahin
den, Immunology Today, 15: 127-133 (1994).
)checking).

In one embodiment, antagonists of CCR1 are identified by monitoring enzyme release upon degranulation or exocytosis by cells that may have this function. Cells expressing CCR1 can be maintained in suitable media under suitable conditions and can induce degranulation. The cells can be contacted with a test agent to assay for enzyme release. Release of the enzyme into the medium can be detected or measured using a suitable assay, such as by an immunological assay or a biochemical assay for enzyme activity.

The media is assayed directly by introducing the assay components (eg, substrates, cofactors, antibodies) into the media (eg, before, concurrently with, or after mixing the cells and agents). be able to. Assays can also be performed on media that has been separated from cells or further processed (eg, fractionated) prior to the assay. For example, convenient assays can be used for enzymes such as serine esterases (eg, for serine esterases from granules, Taub, DD et al., J. Immunol., 155: 3877-38).
88 (1995)).

In another embodiment, the cell expressing CCR1 is a ligand for CCR1 or CC.
The test agent, mixed with a promoter of R1 function, is added before, after or simultaneously with them and degranulation is evaluated. Inhibition of degranulation induced by the ligand or facilitator indicates that the agent is an inhibitor of mammalian CCR1 function.

In a preferred embodiment, the CCR1 function antagonist does not significantly inhibit the function of other chemokine receptors (eg, CCR2, CXCR1, CCR3). Such CCR1 specific antagonists can be identified in any suitable manner, for example, by suitable modifications of the methods described herein. For example, cells that do not express CCR1 (CCR1 ⁻ ) and that express one or more other chemokine receptors (eg, CCR2, CXCR1, CCR3) can be isolated by any suitable method (eg, transfection, antibody staining). , Western blot, RNAse protection). Such cells or cell fractions (eg, membranes) obtained from such cells can be used in a suitable binding assay. For example, CCR1 ^- a is, and if the cell is selected a CCR3 ^+, CCR1 antagonist, cells as described herein, a suitable CCR3 ligand (e.g., RANTES, MCP-3) Alternatively, it can be assayed for its ability to inhibit binding to a cell fraction.

[0052] In another preferred embodiment, the CCR1 function antagonist is an agent that binds to CCR1. Such CCR1 binding antagonists can be labeled in a suitable manner, for example, labeled (eg, enzymatically labeled (eg, alkaline phosphatase, horseradish peroxidase), biotinylated, radiolabeled (eg, ³ H, ¹⁴ C, ¹²⁵ I)) can be identified in binding assays using antagonists.

In another preferred embodiment, the CCR1 function antagonist is an agent that can inhibit binding of a CCR1 ligand (ie, one or more) to CCR1 (eg, human CCR1).

In a particularly preferred embodiment, a CCR1 functional antagonist is an agent that binds to CCR1 and thereby inhibits binding of a CCR1 ligand (ie, one or more) to CCR1 (eg, human CCR1).

Method of Treatment The term “graft” as used herein refers to an organ and / or tissue obtained from a first mammal, ie, a donor, and which can be transplanted into a second mammal, preferably a human. Say. The term “graft” includes, for example, skin, eyes or parts of the eye (eg, cornea, retina, lens), muscle, bone marrow or cellular components of bone marrow (eg, stem cells, progenitor cells),
Heart, lung, cardiopulmonary (eg, heart and one lung, heart and both lungs), liver, kidney, pancreas, parathyroid, intestine (eg, colon, small intestine, duodenum), neuronal tissue, bone and vasculature ( For example, arteries, veins). The graft can be obtained from a suitable mammal (eg, human, pig, baboon, chimpanzee), or under certain circumstances, the graft can be cultured by culturing cells, for example, in a suitable mammal. It can be produced in vitro by culturing embryonic cells, fetal cells, skin cells, blood cells and bone marrow cells obtained from animals. Implants can be obtained from genetically modified animals or can be modified by suitable means (eg, genetically, chemically, physically). The graft is preferably obtained from a human.
As used herein, an "allograft" refers to a graft that contains an antigen that is an allelic variant of the corresponding antigen found in the recipient. For example, HLA-
DRB1 ^* 0401 allele human implant comprising an MHC Class II antigens encoded by is a allografts if the genome is transplanted into a human recipient that does not include the HLA-DRB1 ^* 0401 allele.

In one embodiment, the method of inhibiting (reducing or preventing) graft rejection comprises:
Implant recipients with an effective amount of a CCR1 function antagonist (ie, 1
The above). In another aspect, a method of inhibiting graft rejection comprises administering to an allograft recipient an effective amount of a CCR1 function antagonist. In a preferred embodiment, the method comprises administering to the recipient of the cardiac allograft an effective amount of a CCR1 function antagonist.

In another aspect, the CCR1 function antagonist is a small organic molecule, a natural product,
It is selected from the group consisting of peptides, peptidomimetics and proteins. In this case, the protein is not a chemokine or a variant or analog thereof.

In a preferred embodiment, the present invention provides a recipient of an implant with an effective amount of a CCR.
Provided is a method for inhibiting (reducing or preventing) graft rejection, comprising administering a monofunctional antagonist and an adjuvant therapeutic agent (ie, one or more), preferably an effective amount of an immunosuppressive agent. . Advantageously, the rejection-suppressing effects of CCR1 antagonists and immunosuppressants can be additive or synergistic, and can result in permanent engraftment.

A further benefit of co-administering a CCR1 antagonist and an immunosuppressant is that the dose of immunosuppressant required to inhibit graft rejection can be reduced below the therapeutic level (eg, a single therapeutic agent). (A dose that does not inhibit graft rejection when administered as a). The ability to reduce the dose of the immunosuppressive agent can provide significant benefits to transplant recipients. This is because many immunosuppressants have severe, well-known side effects, such as increased infection morbidity, increased incidence of certain malignancies, Includes incidence, diabetes mellitus, neurotoxicity, nephrotoxicity, hyperlipidemia, hypertension, hirsutism, gingival hyperplasia, impaired healing, lymphopenia, jaundice, anemia, hair loss and thrombocytopenia (Spencer, C. et al. M. et al., Drugs, 54 (6): 92.
5-975 (1997); Physicians Desk Reference.
e, 53rd Edition, Medical Economics Co. Pp. 2081-2
082 (1999)).

As used herein, the term “immunosuppressant” refers to a compound that can suppress an immune response. The immunosuppressant used in the present invention can be a novel compound,
Alternatively, compounds known in the art, for example, calcineurin inhibitors (eg, cyclosporin A, FK-506), IL-2 signaling inhibitors (eg, rapamycin), glucocorticoids (eg, prednisone, dexamethosone) ), Methylprednisolone)
, Nucleic acid synthesis inhibitors (eg, azathioprine, mercaptopurine, mycophenolic acid), and antibodies to lymphocytes or antigen-binding fragments thereof (eg, O
KT3, anti-IL2 receptor). Novel immunosuppressive agents can be identified by those skilled in the art in a suitable manner, for example, by screening compounds for the ability to inhibit antigen-dependent T cell activation.

The immunosuppressive agent used for co-therapy (eg, co-administration with a CCR1 function antagonist) is preferably a calcineurin inhibitor. More preferably, the immunosuppressant used for co-therapy is cyclosporin A.

When the graft is bone marrow, cells (eg, leukocytes) derived from the graft can develop a directed immune response in recipient organs and tissues. Such a condition has been referred to in the art as graft-versus-host disease (GVHD). GVHD can be inhibited by administration of a CCR1 functional antagonist, with or without adjuvant therapeutics (eg, immunosuppressants, hematopoietic growth factors). Thus, in another aspect, the present invention provides a GV in a bone marrow transplant recipient comprising the step of administering an effective amount of a CCR1 function antagonist.
A method for inhibiting (reducing or preventing) HD is provided. In a further aspect, GV
Methods of inhibiting HD comprise a CCR1 function antagonist, one or more adjuvant therapeutic agents,
For example, a step of administering an immunosuppressant is included.

In another aspect, the method of inhibiting GVHD comprises administering a CCR1 functional antagonist selected from the group consisting of a small organic molecule, a natural product, a peptide, a peptidomimetic and a protein. In this case, the protein is not a chemokine or a variant or analog thereof.

A further concept of the invention relates to using a CCR1 antagonist to inhibit ischemia / reperfusion injury. Ischemia / reperfusion injury refers to necrotic cell death that occurs when blood flow to an organ or tissue is restricted or stopped (ischemia) and oxygen deprivation (hypoxia) occurs. Damage to organs or tissues under ischemic conditions appears after blood flow is restored (reperfusion). Ischemia / reperfusion injury can be the result of a pathological condition in which ischemia can occur, such as myocardial infarction, arteriosclerosis, stroke, transient ischemic stroke, and the like. Ischemia / reperfusion injury may also result from trauma or a medical procedure that stops, restricts, or redirects (eg, diverts) blood flow. Examples of such trauma include, for example, frostbite, burns, and pinched injuries that restrict blood flow to the limbs or limbs. Medical procedures that can result in ischemia / reperfusion injury include, for example, placement of tourniquets, angioplasty (eg, balloon angioplasty) and surgery (eg, organ transplantation). In the context of organ transplantation, ischemia / reperfusion injury that occurs upon transplantation of any graft is associated with graft rejection, even in isogenic grafts (where the donor and recipient are identical twins). (Eg, acute rejection, chronic rejection).

A murine model of cold renal ischemia / reperfusion injury that mimics the preservation of an organ (eg, kidney) removed from a donor in preparation for transplantation is disclosed herein (Example 3). checking). Studies using this model have revealed that inhibiting CCR1 function can significantly inhibit ischemia / reperfusion injury. Specifically, renal ischemia / reperfusion had no measurable effect on renal function in CCR1 KO mice, and 100% of the mice survived during the 72 hour follow-up period. Was. In contrast, CCR1 + / + control mice suffered significant impairment of renal function with 80% mortality at 72 hours (Table 3). 4 of ischemia / reperfusion
Histological examination of the resected kidneys at 8 hours post CCR1 KO and CCR1 + / + control mice showed that by inhibiting CCR1 function, necrosis of tubules and infiltration of neutrophils into the kidney were observed. It has been shown that it can be inhibited. Thus, by inhibiting CCR1 function, ischemia / reperfusion injury can be inhibited (reduced or prevented).

Thus, disruption of CCR1 function can have a significant beneficial effect in preventing damage to the transplanted graft (eg, kidney). For example, inhibiting CCR1 function can inhibit (reduce or prevent) early graft damage after transplantation, which can result in reduced acute and chronic allograft rejection. In addition, many organs (eg, kidneys) are preserved for long periods of time resulting in ischemia /
Inhibiting CCR1 function can expand the donor pool of organs because they are not transplanted due to the high risk of reperfusion injury.

Accordingly, another aspect of the present invention is a method for inhibiting ischemia / reperfusion injury, comprising administering to a subject in need thereof (eg, a human) an effective amount of a CCR1 function antagonist. Is the way. In certain embodiments, the ischemia / reperfusion injury may be the result of a medical procedure, trauma, or a pathological condition. In certain aspects, the present invention provides methods for inhibiting ischemia / reperfusion injury that may occur during graft implantation. In a preferred embodiment, the present invention provides a method for inhibiting ischemia / reperfusion injury that may occur during kidney transplantation.

[0068] In another embodiment, the method of inhibiting ischemia / reperfusion injury comprises a CCR selected from the group consisting of small organic molecules, natural products, peptides, peptidomimetics and proteins.
Administering a one-function antagonist. In this case, the protein is not a chemokine or a variant or analog thereof.

In another embodiment, the method of inhibiting ischemia / reperfusion injury comprises administering to a subject in need thereof an effective amount of a CCR1 function antagonist, enhancing blood flow and / or inhibiting leukocyte infiltration. Administering an effective amount of one or more adjuvant therapeutic agents to be obtained.
For example, ancillary agents include thrombotic agents such as fibrinolytic agents (eg, Retavase), plasminogen activators (eg, tissue plasminogen activator, urokinase, streptokinase, recombinant tissue plasminogen activator). Dissolving agents, anticoagulants (e.g., coumarin anticoagulants (e.g., warfarin, ethyledine dicumarol), heparin, hirulog, hirudin, aspirin), vasodilators (e.g., nitroglycerin, amotrifen) (Amotriphene), erythritol, prenilamine), agents that stimulate or inhibit the production of nitric oxide (eg,
Nitric oxide synthase stimulating factor or inhibitor, for example, US Pat.
1,437 (given to Singh et al.), 5,854,234 (given to Hansen et al.) And 5,854,251 (Hallin).
), immunosuppressants and cell adhesion inhibitors. Cell adhesion inhibitors suitable for co-administration with a CCR1 function antagonist include, for example, cytokines, cell adhesion molecules (
For example, antibodies such as integrins and selectins) and antigen-binding fragments thereof, and proteins such as soluble adhesion molecules (eg, chimeric adhesion molecules),
Small organic molecules, peptides and peptidomimetics, for example, US Pat. No. 5,843,4
No. 41 (assigned to Gundel et al.) And US Pat. No. 5,695,760 (F
aanes et al.), US Pat. No. 5,843,425 (assigned to Tedder et al.), US Pat. No. 5,753,617 and US Pat. No. 5,710,123 (
Heavner et al.) And US Pat. No. 5,837,689 (Anderso).
No. 5,725,802 (given to Barrett et al.),
No. 5,510,332 (assigned to Kogan et al.), No. 5,707,9
Nos. 85 and 5,260,277 (assigned to McKenzie et al.), International Patent Applications WO 97/03094, WO 98/04247, WO
96/22966 (Biogen, Inc.), WO 97/10839,
WO96 / 00581 (Texas Biotechnology Cor)
No. WO94 / 15958, WO93 / 08823, and
And the compounds disclosed in WO92 / 08646 (Tanabe Seiyaku Co., Ltd.). The entire teachings of each of the references cited above are incorporated herein by reference.

In a preferred embodiment, the co-agent co-administered with the CCR1 function antagonist may be selected from the group consisting of immunosuppressants and cell adhesion inhibitors. In another preferred embodiment, the ancillary agent may be selected from the group consisting of fibrinolytic agents, thrombolytic agents, anticoagulants, vasodilators, and agents that stimulate or inhibit the production of nitric oxide.

The present invention further provides CCR1 functional antagonists for use in therapy (including prophylaxis), eg, as described herein, and grafts, as described herein. It relates to the use of such antagonists for the manufacture of a medicament for inhibiting rejection (eg acute rejection, chronic rejection) and / or ischemia / reperfusion injury. The present invention also provides a method for inhibiting graft rejection (eg, acute rejection, chronic rejection) and / or ischemia / reperfusion injury.
It relates to a medicament comprising an R1 function antagonist.

A “subject” is preferably a human, but a mammal in need of veterinary treatment, such as a room animal (eg, a dog and cat), a farm animal (eg, a cow, sheep, They can also be chickens, pigs and horses, and experimental animals such as rats, mice and guinea pigs.

An effective amount of a CCR1 functional antagonist is administered in response to graft rejection and / or ischemia /
It can be administered to a subject to inhibit (reduce or prevent) reperfusion injury.
For example, an effective amount of a CCR1 function antagonist can be administered before, during and / or after transplant surgery or other medical procedure that can result in ischemia / reperfusion injury.

When co-administration of a CCR1 function antagonist and an adjuvant therapeutic is indicated or desired to inhibit graft rejection and / or ischemia / reperfusion injury, a CCR1 function antagonist can be It can be administered before, simultaneously with, or after administration of the adjuvant therapeutic agent. If the CCR1 function antagonist and the adjunctive therapeutic are administered at different times, they are preferably the pharmacological activity of the drug (
(Eg, inhibition of CCR1 function, immunosuppression) within a period of time suitable to provide substantial overlap. One of skill in the art can determine the appropriate time for co-administration of a CCR1 function antagonist and adjunctive therapeutic, depending on the particular agent selected and other factors.

An “effective amount” of a CCR1 antagonist is an amount sufficient to achieve the desired therapeutic and / or prophylactic effect and to inhibit graft rejection and / or ischemia / reperfusion injury. Such as a sufficient amount. For example, an effective amount may be a function of CCR1 (ie, one or more) (eg, leukocyte migration induced by CCR1 ligand, integrin activation induced by CCR1 ligand, CCR1 of intracellular free calcium concentration [Ca ²⁺ ] _i . A transient increase induced by the ligand, and / or
Or sufficient to inhibit secretion (eg, degranulation) induced by the CCR1 ligand of a proinflammatory mediator, thereby inhibiting graft rejection and / or ischemia / reperfusion injury Amount. An “effective amount” of an auxiliary therapeutic agent (eg, an immunosuppressant) is an amount sufficient to achieve the desired therapeutic and / or prophylactic effect (eg, immunosuppression).

The amount of drug (eg, CCR1 antagonist, adjunctive therapeutic) administered to an individual may depend on general health, age, gender, weight and resistance to drugs, and rejection and / or ischemia / reperfusion injury Depends on the characteristics of the individual, such as the extent, severity and type. One of skill in the art can determine the appropriate dosage depending on these and other factors. Typically, an effective amount is about 0.1 mg / day for an adult.
It can be in the range of about 100 mg / day. Preferably, the dosage will be from about 1 mg / day to about 1 mg / day.
00 mg / day.

The agents (eg, CCR1 antagonists, adjuvant therapies) can be administered by any suitable route, including, for example, oral administration in a capsule, suspension or tablet, or by parenteral administration. Parenteral administration can include, for example, intramuscular, intravenous, subcutaneous or intraperitoneal administration. Drugs (eg, CCR1
Antagonists, adjuvant therapies) may also be administered orally (eg, in the diet), transdermally or topically, or by inhalation (eg, intrabronchially, intranasally, oral inhalation or intranasal drops) or rectally. can do. Administration can be local or systemic as indicated. The preferred mode of administration depends on the particular agent chosen (eg, CCR
1 antagonist, adjuvant), but oral or parenteral administration is generally preferred.

Agents (eg, CCR1 antagonists, adjuvant therapeutics) can be administered as neutral compounds or salts. Salts of compounds containing an amine or other basic group can be obtained by reaction with a suitable organic or inorganic acid, such as, for example, hydrogen chloride, hydrogen bromide, acetic acid and perchloric acid. Compounds with quaternary ammonium groups also contain counter anions such as chloride, bromide, iodide, acetate and perchlorate. Salts of compounds containing a carboxylic acid group or other acidic functional group can be prepared by reacting with a suitable base, for example, by reacting with a hydroxide base. Salts of the acidic functional groups contain counter cations such as sodium and potassium.

A CCR1 functional antagonist is an individual comprising a CCR1 antagonist and a pharmaceutically acceptable carrier as part of a pharmaceutical composition for inhibiting graft rejection and / or ischemia / reperfusion injury. Can be administered. Pharmaceutical compositions for co-therapy may contain a CCR1 function antagonist and one or more adjuvant therapeutic agents.
The CCR1 function antagonist and the adjuvant therapeutic agent can be components of separate pharmaceutical compositions, which can be mixed together prior to administration, or administered separately. The formulation will depend on the route of administration chosen (eg, solution, emulsion, capsule). Suitable pharmaceutical carriers can include inert ingredients that do not interact with the CCR1 function antagonist and / or adjuvant therapeutic agent. Remington's Pharmaceutical
optical Sciences, Mack Publishing Comp
Standard pharmaceutical compounding techniques can be used, such as those described in Any, Easton, PA. Pharmaceutical carriers suitable for parenteral administration include, for example, sterile water, saline, bacteriostatic saline (saline containing about 0.9% mg / ml benzyl alcohol), phosphate Buffered saline, Hanks's solution, Lactated Ringer's solution and the like are included. Methods of encapsulating the composition (such as with a coating of hard gelatin or cyclodextran) are known in the art (Baker
Et al., "Controlled Release of Biologically Active Agents."
of Biological Active Agents) ", John W.
iley and Sons, 1986).

Next, the present invention will be described with reference to the following examples which are not intended to limit the present invention.

EXAMPLES Example 1 CCR1 Targeting and Heart Transplantation Experimental Design Three sets of data were generated: (I) Acute rejection rate was determined in CCR1 knockout (KO) versus control mice. (Ii) Cyclosporine A (Cs1) versus CCR1 KO versus control mice
The immunosuppressive effect of A) was examined. (Iii) The effect of CCR1 deficiency on the development of chronic rejection was examined.

Methods Mouse. CCR1 KO mice (also called CCR1 − / −) (strain B6 / 129, H-2 ^b ) that are homozygous for target gene disruption of CCR1
aig Gerard (Children's Hospital, Boston)
n, MA; Gerard, C .; J. et al. Clin. Invest. 100: 20
22-2027 (1997)) and LeukoSite (Cam
bridging, Massachusetts). All other mice are available from Jackson Laboratory (Bar Harbor, M.D.).
E). These include donor strain (BALB / c, H-2 d; B6.C-
H2 (bm12) / KhEg (bm12), H-2 ^b ) and control recipient (
B6 / 129). BALB / c and B6 / 129 differ in both major class I and class II major histocompatibility complex (MHC) loci, while Bm1
2 and B6 / 129 differ only in MHC class II.

Allograft of Mouse Heart (Mottram, PL et al., Transplant
nation, 59: 559-565 (1995); W. Proc. Natl. Acad. Sci. (USA), 93: 13967-1.
3972 (1996)) with a surgical microscope (Nikon, 4x-38x magnification).
Was performed under clean conditions with the help of
It was not required for studies involving O mice or control mice, but was required when other immunodeficient mice were used).

Preparation of Donor Heart. Donor mice were treated with Nembutal (50).
mg / 10 g body weight) and atropine sulfate (0.17 mg / 100 g body weight).
p. Anesthetized; additional anesthesia by adding methoxyflurane was given by a facial mask as needed during the procedure. Mice were shaved and cleaned with 70% alcohol. A longitudinal abdominal incision was made in the donor animal and 1 ml of a 10% solution of heparin in saline was injected into the inferior vena cava. Thereafter, the incision was spread cranially and the chest was opened by a median sternotomy. The thorax was opened. The inferior vena cava was ligated with 6-0 silk and separated below the ligature. Thereafter, the superior vena cava was ligated in the same manner and separated above the ligated part. The aorta and pulmonary artery were separated and separated as distally as possible. At this point, blood was drawn from the heart by applying pressure with an applicator stick. The aorta was transected in close proximity to the brachiocephalic artery and the main pulmonary artery was transected in close proximity to its bifurcation. Thereafter, the pulmonary veins were ligated, separated in bulk, and the heart was placed in ice-cold saline.

Preparation of Recipient. After anesthesia in the same manner as the donor, the recipient is carried under a microscope, a longitudinal abdominal incision is made, and the segment of the aorta and vena cava beneath the renal vessels is dissected over a length of about 2 mm. But separated from each other. Clamps were placed in the proximal aorta and vena cava, and distal ligations of 6-0 silk were placed around both the aorta and vena cava in preparation for subsequent vessel closure.

[0086] Heart transplantation. A ligature placed around the distal aorta and vena cava was secured by a single knot. An aortic incision and vena cava incision were performed close to each other. Thereafter, the donor's heart was removed from the chilled saline and the donor's aorta and pulmonary artery were continuously (ru) using 10-0 with a BV-3 needle.
Ninged sutures were laterally joined to the recipient's aorta and vena cava, respectively. Since the anastomosis was performed in close proximity to each other, the pulmonary artery
The side of the vena cava suture was sutured from the inside with continuous valgus suturing. During this time, chilled saline was frequently infused into the ischemic heart. After completing the anastomosis, the ligature closing the lower vessel was first removed, thus filling the inferior vena cava and donor pulmonary artery with the venous blood of the recipient. When the proximal occlusion was released, the aorta and coronary artery of the implant were perfused with oxygenated recipient blood. Blood loss was minimized by gradually releasing the proximal ligature. Warm saline was used externally to warm the heart immediately after establishing coronary perfusion. With warming and coronary perfusion, the heart began to fibrillate and spontaneously returned to sinus rhythm, usually within minutes. Sometimes a heart massage was needed to re-establish a normal heartbeat. The intestine was carefully returned to the peritoneal cavity around the heart substitute, and the abdomen was closed with one continuous suture to all layers (using saline containing antibiotics, The cavity was washed). Thereafter, the mice were placed at a constant temperature of 35 ° C. and allowed to recover from anesthesia.

[0087] Immunosuppression. Cyclosporin A (CsA) (Sigma, St. Louis, M
O) The effect of therapy (10 mg / kg / day, subcutaneous injection) was determined by comparing BALB / c-> B6 /
129 or B6 / 129-> CCR1 KO using our standard cardiac allograft model by daily injection of CsA until rejection appears or for up to 21 days starting on the day of transplantation Examined.

Monitoring allograft survival. Survival of cardiac allografts is determined by palpation of ventricular contractions through the abdominal wall (Mottram, PL et al., Transplantation).
, 59: 559-565 (1995)). Rejection was defined as the palpable cessation date of the beat and was confirmed by necropsy (Ge
rad, C.I. J. et al. Clin Invest. , 100: 2022-202
7 (1997); Motram, P .; L. Et al., Transplantatio
n, 59: 559-565 (1995)). If the heart graft stops functioning,
Mice are anesthetized as described above, and the grafts are surgically excised and (a) formalin-fixed, paraffin-embedded and subsequently optical microscopy, or (b) snap frozen in liquid nitrogen for immunohistology or RNAse. Aliquots were stored for storage at -70 ° C until processed for protection assays.

[0089] Immunopathology. For histology, paraffin sections were stained with hematoxylin and eosin (H & E) to assess graft morphology and stained with Weigert's elastin stain for intimal proliferation at the ingress of the myocardial artery (transplant (An important feature of atherosclerosis in rats) (Gerard, C. et al., JC).
lin Invest. , 100: 2022-2027 (1997); Mott.
ram, P .; L. Et al., Transplantation, 59: 559-565.
(1995)). Chemokine and chemokine receptor mRNA expression is
Measured using a protection assay kit (Pharmingen, San Diego, CA).

Results Allograft survival data (mean ± SD) are summarized in Table 1 (6 to 1 animal per group)
0 animals are used).

[0091]

[Table 1]

Important points from Table 1 are as follows: CCR1 ⁺ cells contribute to the pathogenesis of allograft rejection.

Disruption of CCR1 function in a complete MHC mismatch significantly prolongs allograft rejection (group 1 vs. 2). As expected, rejection in control mice was associated with graft infiltration by CCR1 ⁺ mononuclear cells (primarily macrophages). In addition, mRNA studies showed that rejection in control mice
It has been shown to be associated with intragraft induction of mRNA expression for R1 ligand, MIP-1α and RANTES. Rejection in CCR1 KO mice was associated with confluent mononuclear cell infiltration and upregulation of CCR1 ligand mRNA, but allografts had no CCR1 mRNA or protein expression.

The addition of CsA (10 mg / kg / d) only slightly increased allograft survival in control mice (2-3 days) when compared to untreated recipients (group 1: 3). However, the same dose of CsA in CCR1 KO mice (up to 21 days) resulted in permanent engraftment in all recipients (Group 4, by further evaluation, Group 4 mice had> 20
Survived over 0 days). The beneficial effects of some experimental drugs may be attenuated by concomitant immunosuppression. However, Cs
Inhibition of A and CCR1 function is synergistic in potency. In addition, grafts harvested at day 100 from CCR1 KO recipients showed only minimal mononuclear cell infiltration and no evidence of graft arteriosclerosis. These findings are in contrast to the severe arteriosclerosis observed in control allograft recipients treated with high dose CsA (30 mg / kg / d) or CD4 mAb treatment (Mottram, P. L. Han, WR et al., "Short-term CD4.
I in cardiac allografts of long-term surviving mice after monoclonal antibody treatment
Increased expression of L-4 and IL-10 and decreased expression of IL-2 and IFN-γ ", Transplantation, 59: 559-565 (1995).
Hancock, W .; W. Buelow, R .; "Antibody induced graft arteriosclerosis is prevented by graft expression of antioxidant and anti-apoptotic genes", Nature Medicine, 4: 1392-1396.
(1998)).

[0095] Disruption of CCR1 function in a combination of class II mismatches is very effective. Untreated recipients rejected the allograft at about day 35 (group 5), but disruption of CCR1 function in this combination resulted in permanent engraftment (group 6). Moreover, as in group 4 (low dose CsA), 10
Allografts taken on day 0 showed no evidence of graft atherosclerosis or other features of chronic rejection.

Example 2 CCR1 and Chronic Rejection in Cardiac Allograft Recipients Administration of a CD4 monoclonal antibody (mAb) can prolong the survival of cardiac allografts in the described murine model (Mottram et al. ,
Transplantation, 59: 559-565 (1995)). However, the prolonged survival of grafts in animals treated with anti-CD4 is complicated by the development of chronic rejection with symptomatic graft arteriosclerosis (Hanco
ck et al., Nature Medicine 4: 1392-1396 (1998).
)).

Methods Cardiac allografts from Balb / c donors were implanted into CCR1 KO mice or CCR1 + / + control mice as described in Example 1.

[0098] Immunosuppression. CD4 mAb (GK1.5, American Type Cu
lture Collection, Manassas, VA; registration number TIB
-207) was administered four times to CCR1 − / − or CCR1 + / + allograft recipients (6 per group); day 0 (at the time of transplantation) and subsequent days 1, 2, and 3 250 μg by intraperitoneal injection into eyes.

Monitoring of chronic rejection. Heart allograft survival was monitored twice daily by palpation of ventricular contractions through the abdominal wall. All grafts survived up to 60 days. The reading was then morphological, especially the degree of onset of graft arteriosclerosis. Thus, the graft is fixed in formalin, embedded in paraffin, and
Eigert was counterstained with elastin staining. All intramyocardial arteries were <5% occluded (0),> 5% -20% (1),> 20%-
40% (2),> 40% to 60% (3),> 60% to 80% (4), or> 80
% To 100% (5) (Murphy et al., Transplanta).
, 64: 14-19 (1997)).

Results The results and the statistical evaluation (Mann-Whitney U test) of the blood vessels in the heart allograft (6 grafts per group) are shown in Table 2.

[0101]

[Table 2]

Important points from Table 2 include: CCR1 ⁺ cells contribute to the pathogenesis of chronic allograft rejection.

Disruption of CCR1 function prevents the onset of atherosclerosis in the implant.

Disruption of CCR1 function prevents the development of other features of chronic rejection.

Example 3 CCR1 and Renal Ischemia / Reperfusion Injury Prolonged interruption of blood supply to an organ leads to its complete necrosis even when the circulation is finally restored. Shorter periods of interruption can result in the death of certain cell types that are highly susceptible to hypoxia, but the entire organ remains partially dependent on the period of blood loss (ischemia) and other factors. It can recover to a target or to a complete extent. The damage that appears after the blood flow to the primary ischemic kidney or other commonly transplanted organs is restored is also known as ischemia / reperfusion injury, It is mainly mediated by neutrophils that gather in Although a number of pathways are involved in the recruitment of such neutrophils, we believe that blocking this pathway will improve the chemokine receptor CCR1 so that ischemia / reperfusion is ameliorated. Has hypothesized that may play an important role. The present inventors found that 4 organs during the ischemic period
Maintaining at 0 ° C. established a new mouse model of cold kidney ischemia / reperfusion injury that mimics the storage conditions of the kidneys removed in preparation for transplantation. Thus, these results are directly related to the method described herein, ie, the method by which clinical ischemia / reperfusion injury in renal allografts can be reduced or even eliminated by blocking the CCR1 pathway. .

Experimental Design Inbred CCR1 KO mice or control B6 / 129 kidneys were perfused in situ using cold saline until white and in ice for various periods until revascularization. Sealed. After preliminary studies, we performed 60 minutes of cold ischemia and 48-72 hours of follow-up to assess renal function, histology, immunopathology (by quantitative image analysis), and mRNA analysis. Done.

Methods Mouse. CCR1 KO mice (B6 / 129 strain, H-2 ^b )
g Gerard (Children's Hospital, Boston,
MA; Gerard, C.M. J. et al. Clin. Invest. 100: 2022
-2027 (1997)) and LeukoSite (Cambr)
id, Massachusetts). Control B6 / 1
Twenty-nine mice were transferred to Jackson Laboratory (Bar Harbor,
ME).

The ischemia / reperfusion model. The ischemia / reperfusion procedure was performed using a surgical microscope (Zeiss, 2 ×
Performed under sterile conditions with the help of 〜50 × magnification). Mice (CCR1 KO or B6 / 129) were anesthetized with Nembutal (50 mg / 100 g body weight); additional methoxyflurane administered via a facial mask was used to provide additional anesthesia if needed. After shaving and disinfecting the abdomen with 70% alcohol, laparotomy was performed by longitudinal abdominal incision. After reaching the renal artery and renal vein of the right kidney, the suprarenal and subrenal vena cava and aorta were separated, the tissues around the kidney were cut, and the kidney was moved. In order to stop arterial blood flow to the kidney, the aorta of the subrenal and epithelium was closed by applying a microclip. After that, an aortic incision (
aortomy) and the kidney was flushed with cold saline until white.
A small incision was made in the renal vein between the clip and the kidney after closing the vein with a microclip to allow reflux of the lavage fluid in the abdomen. All incisions were sutured with 10-0 prolene suture. After this step,
Kidneys were exposed to various ischemic times up to 75 minutes. During this time, the kidney was kept cool by placing it on ice.

Following removal of the clips and reperfusion of the organ, a contralateral nephrectomy was performed. 6-
0 closed with silk thread. The animals were kept in a warmed cage with observation after surgery until it was noted that they had recovered from the procedure.

Monitoring the effects of ischemia / reperfusion injury. Serum creatinine levels were measured daily. Since creatinine levels reached a maximum 48 hours after ischemia / reperfusion,
Mice (8 per group) were used for 48 for kidney histological and mRNA studies.
They were sacrificed at time or watched for 72 hours to determine their effect on animal survival. For histology, kidneys were fixed in formalin, embedded in paraffin, and stained with hematoxylin and eosin (H & E) and periodic acid / Schiff (
(PAS) staining. Chemokines and chemokine receptor mRNA expression
NAse protection assay kit (Pharmingen, San Diego, C
Measured using A).

Results Contrasting findings in control B6 / 129 mice versus CCR1 mice are shown in Table 3.

[0112]

[Table 3]

Important points from Table 3 are as follows: Disruption of CCR1 function prevented recruitment of neutrophils after ischemia / reperfusion injury and completely prevented renal dysfunction . CCR1 blockade also almost completely suppressed tubular cell necrosis.

Table 3 does not show the results of mRNA analysis. CCR1 KO and B6 /
Kidneys from both 129 mice were CCR1-related chemokines, MIP-1α.
And RANTES induction of mRNA expression. Thus, the effect of CCR1 deficiency did not impair the pro-inflammatory cellular response, but the absence of CCR1 in the host cells suppressed leukocyte recruitment and subsequent renal injury.

The study was expanded by increasing the number of animals in each group to 15 and extending the analysis period to 100 days. The results shown in Table 3 were included in the analysis of this extended study. According to an expanded study, CCR1 − / − mice show ischemic /
After reperfusion, it was shown to survive significantly longer than B6 / 129 mice (9
2 ± 6.1 days vs 45 ± 12.4 days, mean survival ± standard error of the mean, limited to 100 days, p <0.001). 86% of the CCR1 − / − mice were still alive 100 days after the ischemia / reperfusion procedure, whereas B6 / 129 mice only 26% survived 100 days after the procedure (FIG. 1). Serum creatinine measurements were taken at 2, 7, and 100 days after the ischemia / reperfusion procedure to assess renal function. CCR
1-/-mice were protected from renal injury and showed serum creatinine levels similar to those detected in sham animals (one-sided nephrectomy, no ischemia / reperfusion). However, serum creatinine levels were significantly increased by about 5-fold in wild-type B6 / 129 animals 2 days after ischemia / reperfusion (p <0.
005). A significantly elevated (4-fold) serum creatinine level was also detected in wild-type B6 / 129 animals 7 days after ischemia / reperfusion (p <0.005).
Serum creatinine was elevated (p <0.01) in all groups, including sham-treated animals, after 100 days of the procedure (FIG. 2).

Example 4 CCR1 and Liver Ischemia / Reperfusion Injury Methods Male C57B6 / 129 (B6 / 129) or male CCR1 − / − mice weighing 22-28 g were used. Partial liver ischemia was induced in mice anesthetized with sodium pentobarbital (60 mg / kg ip). A midline laparotomy was performed and atraumatic clips were used to cut off the blood supply to the middle and left lobes of the liver, thereby creating partial hepatic ischemia. After 90 minutes, the clips were removed and liver reperfusion was started. Sham-operated control mice received the same surgical procedure without interrupting the blood supply to the middle and left lobes of the liver. Mice were sacrificed at a series of intervals, and liver tissue and blood samples were collected for analysis. Liver function was assessed by measuring serum alanine aminotransferase (SGPT) and / or serum aspartate aminotransferase (SGOT).

Results CCR1 − / − mice were tested for serum alanine aminotransferase (SGPT).
0.5) after liver ischemia / reperfusion as assessed by (FIG. 3) and serum aspartate aminotransferase (SGOT) assays.
She was protected from liver dysfunction for 7 days. In addition, CCR1-/-mice are
After hepatic ischemia / reperfusion, they survived significantly longer than B6 / 129 wild-type mice (13.4 ± 3.6 days compared to 8.7 ± 5.1 days, p <0. 01). Immunohistological analysis of liver tissue revealed CCR1 ligand expression induced by ischemia / reperfusion in CCR1-/-and B6 / 129 mice. Extensive liver necrosis and neutrophil infiltration indicate that B after liver ischemia / reperfusion
It was found in liver tissue removed from 6/129 mice. However, CCR1 − / − mice after hepatic ischemia / reperfusion showed only slight localized hepatocellular injury and no neutrophil infiltration. Therefore, CC
The absence of R1 suppressed liver ischemia / reperfusion injury and subsequent liver dysfunction.

Example 5 Immune Competence of CCR1 − / − Mice In Vitro T Cell Proliferative Response Mixed lymphocyte response (MLR) was determined by using pancreatic cells (CCR1 − / − or wild-type B6 / 129 mice) of responding animals. Isolated) with pancreatic cells (isolated from Balb / c mice) of allogeneic stimulator cells inactivated with mitomycin C, 5% FBS, 1%
Evaluated by culturing in 96-well flat bottom plates in RPMI-1640 medium containing penicillin / streptomycin and 5 × 10 ⁻⁵ M 2-mercaptoethanol. Cultures were incubated at 37 ° C. with 5% CO ₂ for 3-5 days and pulsed with [ ³ H] thymidine for 6 hours before harvesting.
The amount of [ ³ H] thymidine taken up by the cultured cells was determined by scintillation counting. The average amount of radioactivity incorporated (counts per minute) and standard deviation were calculated using 12 wells per group.

The mitogen-induced T-cell proliferation was determined by concanavalin A (Con-A
, T cell mitogen). CCR1 − / − mouse or wild type B
Pancreatic cells isolated from 6/129 mice were treated with 5% FBS, 1% penicillin / streptomycin, 5 × 10 ⁻⁵ M 2-mercaptoethanol, and 1.25 μg
/ ML to 10 μg / mL of Con-A (Sigma Chemical Co.).
, St. (Louis, MO) in 96-well flat bottom plates in RPMI-1640 medium. Cultures were incubated at 37 ° C. with 5% CO ₂ for 72 hours and pulsed with [ ³ H] thymidine for 6 hours before harvesting.
The amount of [ ³ H] thymidine taken up by the cultured cells was determined by scintillation counting. The average amount of radioactivity incorporated (counts per minute) and standard deviation were calculated using 12 wells per group.

Results The results of the in vitro T cell proliferation study are shown in FIGS. 4A and 4B. CCR1-
<RTIgt; /-mitogen-induced </ RTI> T cell proliferation detected in cultures of pancreatic cells obtained from mice was shown to be mitogen-induced T cells detected in cultures of pancreatic cells obtained from wild-type B6 / 129 mice. It was identical to proliferation (FIG. 4A). CCR1 − / − mice showed a violent response to allogeneic stimulator cells in the MLR assay (p <0.001) (FIG. 4B). However, the overall magnitude of the response in the repeated 5-day MLR assay showed that the responder animal pancreatic isolated from CCR1 − / − mice compared to cultures containing B6 / 129 responder pancreatic cells. Cultures containing cells were consistently 20% -25% lower (p <0.01). These studies of in vitro T cell responses reveal that CCR1 − / − mice are immunoreactive.

While the invention has been particularly shown and described with reference to preferred embodiments thereof,
It will be understood by those skilled in the art that various changes in form and detail may be made in the present invention without departing from the spirit and scope of the invention as defined by the appended claims.

[Brief description of the drawings]

FIG. 1. CCR1 − / − mice (− ◇ −) after renal ischemia / reperfusion.
And Kaplan-Meier survival curves illustrating the survival of wild-type B6 / 129 mice (-２９-). CCR1 − / − mice survived significantly longer than B6 / 129 mice (p <0.001, log rank sum test, survival limited to 100 days).

FIG. 2 shows sera of CCR1 − / − and B6 / 129 mice after renal ischemia / reperfusion or sham surgery (one-sided nephrectomy without ischemia / reperfusion). 4 is a histogram illustrating creatinine levels. Creatinine levels are reduced by ischemia /
Measured at 2, 7, and 100 days after reperfusion or sham operation. B6
Only / 129 mice (control) had significantly elevated serum creatinine levels 2 and 7 days after ischemia / reperfusion ( ^** p <0.005). All groups had elevated levels of serum creatinine 100 days after ischemia / reperfusion or sham surgery ( ^* p <0.01).

FIG. 3 shows CCR1 − / − (K) at a predetermined time after liver ischemia / reperfusion
O) Histogram illustrating serum alanine aminotransferase (SGPT) levels as indicators of liver dysfunction in mice and B6 / 129 (WT) mice. B6 / 12 who underwent surgery but did not experience hepatic ischemia / reperfusion
SGPT levels in the serum of 9 mice (sham operation) are also shown. CCR
1-/-mice have significantly lower liver dysfunction than B6 / 129 mice, as assessed by serum SGPT levels over a period of 0.5 to 7 days following hepatic ischemia / reperfusion. ( ^* P <0.0001).

FIG. 4A shows CCR1 − / − or B6 / 129 mice (CCR1 + / +)
4 is a histogram illustrating the concanavalin A-induced proliferation of T cells in cultures of pancreatic cells isolated from. The T cell proliferative responses induced by concanavalin A in cultures of cells obtained from CCR1 − / − mice and cultures of cells obtained from B6 / 129 mice were approximately equivalent. Data are the mean ± SD of 6 cultures that were stimulated for 48 hours. Data shown is 4
Represents an assay.

FIG. 4B shows CCR1 − / − mice (CCR1 KO) or wild-type B6 / 129 mice (WT) stimulated with allogeneic pancreatic cells (isolated from Balb / c mice).
4 is a histogram illustrating the mixed lymphocyte response (MLR) of the cells isolated from (a). Cells obtained from both CCR1-/-and wild-type B6 / 129 mice were:
Demonstrated robust MLR against Balb / c stimulated cells treated with mitomycin C ( ^** p <0.001, unstimulated CCR1 − / − or unstimulated wild-type B6 /
129 responder cells). CCR1 − / − cells grew less than wild type cells in the assay ( ^* p <0.01). Data are means ± SD of six 5-day cultures. The data shown represents four assays. BA
LB / c stim. : Pancreatic cells isolated from Balb / c mice and treated with mitomycin C; WT resp. : Pancreatic cells isolated from B6 / 129 mice; CCR1 KO resp. : Pancreatic cells isolated from CCR1-/-mice; BALB / c-> WT: Isolated from B6 / 129 mice isolated from Balb / c mice and stimulated with mitomycin C-treated pancreatic cells. Pancreatic cells; BA
LB / c-> KO: Pancreatic cells isolated from CCR1 − / − mice isolated from Balb / c mice and stimulated with mitomycin C-treated pancreatic cells.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 39/395 A61K 45/06 45/06 A61P 9/10 A61P 9/10 37/06 37/06 A61K 37 / 02 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF , BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ) , UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, A, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE , KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZWF terms (reference) 4C084 AA01 AA02 AA03 AA17 AA20 BA05 BA44 CA62 MA13 MA17 MA22 MA23 MA31 MA35 MA36 MA37 MA41 MA43 MA52 MA59 MA60 MA66 NA14 ZA362 ZA542 ZB082 ZC202 ZC412 ZC422 ZC752 4C085 AA13 AA14 BB11 CC02 CC12 CC23 CC32 DD88 EE01 EE03 FF24 GG02 GG02 A04 MA02 MA04 MA13 MA17 MA22 MA23 MA28 MA31 MA35 MA36 MA37 MA41 MA43 MA52 MA56 M A59 MA60 MA63 MA66 NA14 ZA36 ZA54 ZB08 ZC41 ZC75

Claims (48)

[Claims] 1. A method for inhibiting transplant rejection, comprising: administering an effective amount of a CCR1 function antagonist to a subject in need thereof. 2. The method of claim 1, wherein said implant is an allograft. 3. The method according to claim 1, wherein the allograft is a kidney, liver, lung, heart-lung (heart-lu
3. The method of claim 2, wherein the method is selected from the group consisting of ng), pancreas, intestine, and heart. 4. The method of claim 3, wherein said allograft is a heart. 5. The method of claim 5, wherein the CCR1 function antagonist is a small organic molecule, a natural product,
Peptides, proteins and those selected from the group consisting of peptide mimetics,
The method of claim 1. 6. The method of claim 5, wherein said CCR1 function antagonist is a small organic molecule. 7. The method of claim 5, wherein said CCR1 function antagonist is a natural product. 8. The method of claim 5, wherein said CCR1 function antagonist is a peptide. 9. The CCR1 function antagonist is a peptidomimetic,
The method of claim 5. 10. The method of claim 5, wherein said CCR1 function antagonist is a protein. 11. The method according to claim 10, wherein said protein is an anti-CCR1 antibody or an antigen-binding fragment thereof. 12. A method for inhibiting transplant rejection, comprising administering to a subject in need thereof an effective amount of a CCR1 function antagonist and an immunosuppressant. 13. The immunosuppressive agent is one or more agents selected from the group consisting of calcineurin inhibitors, glucocorticoids, nucleic acid synthesis inhibitors, antibodies that bind to lymphocytes, and antigen-binding fragments of the antibodies. 13. The method of claim 12, wherein 14. The method of claim 13, wherein said immunosuppressant is a calcineurin inhibitor. 15. The method according to claim 14, wherein said calcineurin inhibitor is cyclosporin A. 16. The calcineurin inhibitor is FK-506.
The method of claim 14. 17. The method of claim 13, wherein said immunosuppressant is a glucocorticoid.
The described method. 18. The method of claim 17, wherein said glucocorticoid is prednisone or methylprednisolone. 19. A method for inhibiting graft-versus-host disease, comprising administering to a recipient of a transplanted graft an effective amount of a CCR1 function antagonist. 20. The method of claim 19, wherein said graft is bone marrow. 21. The method of claim 20, further comprising administering an immunosuppressant.
The described method. 22. The method of claim 21, wherein said immunosuppressant is a calcineurin inhibitor. 23. The method of claim 22, wherein said calcineurin inhibitor is cyclosporin A or FK-506. 24. A method of inhibiting ischemia / reperfusion injury, comprising administering to a subject in need thereof an effective amount of a CCR1 function antagonist. 25. The method of claim 24, wherein said CCR1 function antagonist is selected from the group consisting of small organic molecules, natural products, peptides, proteins and peptidomimetics. 26. The method of claim 25, wherein said CCR1 function antagonist is a small organic molecule. 27. The method of claim 25, wherein said CCR1 function antagonist is a natural product. 28. The method of claim 25, wherein said CCR1 function antagonist is a peptide. 29. The method of claim 25, wherein said CCR1 function antagonist is a peptidomimetic. 30. The method of claim 25, wherein said CCR1 function antagonist is a protein. 31. The method according to claim 30, wherein said protein is an anti-CCR1 antibody or an antigen-binding fragment thereof. 32. The method of claim 25, wherein the ischemia / reperfusion injury is the result of a medical procedure that stops, restricts, or redirects blood flow. 33. The medical procedure is angioplasty or surgery.
The described method. 34. The method of claim 32, wherein said medical procedure is surgery. 35. The method of claim 34, wherein the surgery is a graft transplant. 36. The method of claim 35, wherein said graft is selected from the group consisting of kidney, lung, liver, cardiopulmonary, pancreas, intestine and heart. 37. The method of claim 36, wherein said implant is a kidney. 38. The ischemia / reperfusion injury is a result of a pathological condition selected from the group consisting of arteriosclerosis, myocardial infarction, stroke and transient ischemic stroke.
The described method. 39. The method of claim 38, wherein said pathological condition is myocardial infarction. 40. The method of claim 38, wherein said pathological condition is stroke. 41. The method of claim 38, wherein said pathological condition is arteriosclerosis. 42. The subject may be provided with a fibrinolytic agent, thrombolytic agent, anticoagulant,
25. The method of claim 24, further comprising administering an effective amount of one or more adjuvant therapeutic agents selected from the group consisting of a cell adhesion inhibitor, an antithrombotic agent, a nitric oxide synthase stimulating factor, and a nitric oxide synthase inhibitor. Method. 43. The method of claim 42, wherein said adjunctive therapeutic is a fibrinolytic agent. 44. The method of claim 42, wherein said adjuvant therapeutic agent is a thrombolytic agent. 45. The method of claim 42, wherein said adjuvant therapeutic is an anticoagulant. 46. The method of claim 4, wherein the adjuvant is a cell adhesion inhibitor.
2. The method according to 2. 47. The method of claim 42, wherein said adjuvant is an antithrombotic agent. 48. The method of claim 42, wherein said adjunctive therapeutic is a nitric oxide synthase activator or a nitric oxide synthase inhibitor.