WO1997021344A1 - Pathologic animal having pleurisy - Google Patents

Abstract

The present invention provides a novel pathologic animal having pleurisy and a method for preparation thereof. Said pathologic animal is prepared by administration of lipoteichoic acid, and useful for screening a substance for preventing or treating inflammation such as pleurisy.

Description

DESCRIPTION PATHOLOGIC ANIMAL HAVING PLEURISY BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to a pathologic animal having pleurisy prepared by administration of lipoteichoic acid, a method for preparation thereof, and a method for assessment of medicines using said animal.

In screening tests of various substances for their pharmacological activities, particularly for the purpose of developing a medicine, a pathologic animal is preferably used rather than a normal animal. The reason is that the pathologic animal, in general, develops symptoms resembling those clinically observed in human, and therefore, such animal is convenient for assessment of medicines. Related Art Statement Pleurisy is an acute inflammation leading to an extensive disorder of the pleura, which is caused by incursion into blood circulation of endotoxins, tissue debris, or various harmful substances. The disease includes sclerotic pleurisy, suppurative pleurisy, pleurisy with effusion, proliferative pleurisy and cystic pleurisy, etc., and prevention, treatment, and diagnosis of this disease at early stage are large medical concerns.

It has recently been reported that E-selectin which is a neutrophil- adhesive molecule expressed on vascular endothelial cells, P-selectin which is a neutrophil-adhesive molecule expressed on vascular endothelial cells and platelets, and L-selectin which is a homing receptor of lymphocytes, recognize Lewis X and sialyl Lewis X sugar chain structures as a ligand (Shigeaki MOROOKA,"lgakunoayumi" (J. Clin. Exp. Med.), 169:108 (1994)). For example, onset of various inflammatory diseases is initiated by interaction between the selectins and their ligands, which results in their binding, and therefore, a substance inhibiting such adhesion is expected to be a possible anti-inflammatory drug (M.P.Bevilacqua et al., Thrombosis Haemostasis, 70:

152 (1993)). Accordingly, sugar chain derivatives or antibodies to various selectins are expected to be applicable for the treatment of selectin-related diseases, and attempts are being made to use the derivatives or antibodies as therapeutic agents for the diseases. As in vivo pathologic models wherein anti-inflammatory activity of selectin inhibitors was observed, IgG immune complex (M.S. Mulligan et al., J. Exp. Med., 178: 623 (1993)), lung disorder induced by cobra venom factor (M. S. Mulligan et al., Nature, 364: 149 (1993)), or reperfusion injury after cardiac ischemia (D. Letter et al., J. Clin. Invest., 93: 1140 (1994)), etc. are known. A pathologic animal used for screening of a medicine for treating pleurisy has been known for a long time (for example, W. G. Spector and D. A. Willoughby, The Pharmacology of Inflammation, The English Univ. Press (1968)). To induce pleurisy, silver nitrate, carrageenin, kaolin, turpentine oil and croton oil, etc., as well as gum arabic, have been used (C. G. Van Arman, Handbook of Experimental Pharmacology, Vol. 20, Anti-inflammatory Drugs, p.75-91 , Springer (1979)), and, for pathologic animals, rats, mice, guinea pigs and rabbits are used. However, application of selectin inhibitors to a therapy of pleurisy has not been tried, for the reasons of lack of a method for evaluating the inhibitors for neutrophil function using a suitable pathologic animal having pleurisy. SUMMARY OF THE INVENTION A purpose of the present invention is to provide a new pathologic animal having pleurisy, a method for preparation thereof and a method for evaluation of selectin inhibitors using said animal.

The present inventor has found that a pathologic animal having pleurisy can be prepared by administering lipoteichoic acid to the animal. The present invention is based on such findings and additional studies based on such findings. Purposes of the present invention are to provide:

(1 ) A pathologic animal having pleurisy, which is prepared by administration of lipoteichoic acid.

(2) The pathologic animal described in the above item (1 ), wherein the onset of pleurisy is mediated by a selectin. (3) The pathologic animal described in the above item (2), wherein the selectin is E-selectin.

(4) A method for preparing a pathologic animal having pleurisy characterized by administrating lipoteichoic acid to an animal.

(5) The method for preparing a pathologic animal described in the above item (4), which method is characterized by the onset of pleurisy mediated by a selectin.

(6) The method for preparing a pathologic animal described in the above item (5) wherein the selectin is E-selectin.

(7) A method for evaluating a medicine for preventing or treating inflammation, which comprises using a pathologic animal having pleurisy prepared by administration of lipoteichoic acid to an animal.

(8) The method for evaluating a medicine described in the above item (7), wherein the medicine is a selectin inhibitor.

(9) The method for evaluating a medicine described in the above item (8), wherein the selectin inhibitor is an E-selectin inhibitor.

(10) A method for screening a medicine for preventing or treating inflammation, which comprises the steps of:

(a) administering a chemical substance to be tested to a pathologic animal having pleurisy prepared by administration of lipoteichoic acid to an animal; and

(b) determining ability of said substance for treating or preventing pleurisy in said pathologic animal.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 :

Figure 1 represents therapeutic effects of anti-rat E-selectin monoclonal antibody on pleurisy caused by the administration of lipoteichoic acid. The vertical axis shows the number of neutrophils infiltrated into the pleural cavity. The administration of lipoteichoic acid (0.1 mg/kg) into pleural cavity in rats induced significant amount of neutrophil accumulation into pleural cavity, which was significantly inhibited by administration of anti-rat E-selectin monoclonal antibody (0.5 mg/kg, 1.0 mg/kg and 2.0 mg/kg) in a dose- dependent manner. Figure 2: Figure 2 represents therapeutic effects of dexamethasone on pleurisy caused by the administration of lipoteichoic acid. The vertical axis shows the number of neutrophils infiltrated into the pleural cavity. The administration of lipoteichoic acid (0.1 mg/kg) into pleural cavity in rats induced significant amount of neutrophil accumulation into pleural cavity, which was significantly inhibited by administration of dexamethasone (0.5 mg/kg).

Figure 3:

Figure 3 represents therapeutic effects of sialyl Lewis X derivative on pleurisy caused by the administration of lipoteichoic acid. The vertical axis shows the number of neutrophils infiltrated into the pleural cavity. The administration of lipoteichoic acid (0.1 mg/kg) into pleural cavity in rats induced significant amount of neutrophil accumulation into pleural cavity, which was significantly inhibited by administration of sialyl Lewis X oligosaccharide (30 mg/kg).

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be described in detail below. For pathologic animals of the present invention, there can be used animals conventionally and frequently used in a laboratory, such as mice, rats, guinea pigs, hamsters, rabbits, dogs and monkeys. Lipoteichoic acid is a representative polymer existing on the surface of

Gram-positive bacteria, which constructs a high-molecular structure via the diester phosphate moiety, and it is known that the acid plays an important role on the onset of sepsis, pneumonia or the like (M. J. How et al., Adv. Carb. Chem., 19: 303-358 (1964)). Bacteria, which are source of lipoteichoic acid used for preparing the pathologic animal of the present invention, include ones which are easily available, such as Staphylococcus aureus (J.Baddiley et al., Biochem. J. 85: 49 (1962)), Bacillus subtilus (W. R. de Boer et al., Eur. J. Biochem, 62: 1 (1976)), or the like.

Treatment of human umbilical vein endothelial cells with lipoteichoic acid has led to the expression of E-selectin on the vascular endothelial cells in a dose- and time-dependent manner. The stimulated endothelial cells has caused adhesion of neutrophils added, depending on the amount of E- selectin expressed. The adhesion of neutrophils was remarkably inhibited by an anti-E-selectin antibody and a certain oligosaccharide derivative which is a ligand of E-selectin.

On the preparation of a pathologic animal having pleurisy of the present invention, a single dose administration of lipoteichoic acid can induce the onset of pleurisy, while divided administrations of a small amount of the acid may also be effective. Intrapleural administration is a preferable administration route. An optimum dose of lipoteichoic acid can be determined in conventional manner depending on the species, strains, sex, age (week old), or fasting conditions of animals, etc.. For example, the pathologic animal can be prepared by administration of lipoteichoic acid into pleural cavity in rats or mice at a single dose of 0.01 - 1 mg/kg, preferably 0.1 mg/kg. It was confirmed that administration of lipoteichoic acid into pleural cavity in a laboratory animal has caused neutrophil accumulation into the pleural cavity followed by the onset of pleurisy. Such neutrophil accumulation induced by lipoteichoic acid was significantly inhibited by an anti-E-selectin antibody in a dose-dependent manner. The result suggested that the onset of pleurisy by administration of lipoteichoic acid was mediated by selectin. Accordingly, representative examples of substances which can be screened using a pathologic animal having pleurisy of the present invention include a selectin inhibitor, particularly an E-selectin inhibitor. Selectin inhibitors prevent an action of selectin as a cell adhesion molecule in vivo. Selectin inhibitors include ligands for selectin and derivatives thereof, antibodies specific to the ligand, biosynthetic inhibitors for the selectin ligand, anti-selectin antibodies, selectins and fragments thereof, and inhibitors for selectin expression.

Ligands for selectin and derivatives thereof include leukocyte-surface glycoproteins, glycolipids, oligosaccharides which constitute a terminal structure of them, and derivatives thereof. For example, oligosaccharides and derivatives thereof include sialyl Lewis X and sialyl Lewis X derivatives, Lewis X and Lewis X derivatives, sulfated sugar and phosphated sugar, etc. (see, for example, Varki et al., Proc. Natl. Acad. Sci. USA. 91 : 7390 (1994)). Examples of glycoproteins include PSGL-1 (see, for example, Sako et al., Cell. 75: 1179

(1993)).

An antibody specific to ligand for selectin means an antibody specific to the foregoing ligands. The antibody may be either a polyclonal antibody or a monoclonal antibody. The antibody can originate from any species and examples of such antibodies include mice- or human-derived antibodies, chimera antibodies between human and mice, or humanized antibodies, etc.. Specifically, anti-sialyl Lewis X antibody, anti-sialyl Lewis a antibody, anti- Lewis X antibody or anti-Lewis a antibody, etc. are included, (see, for example, Fukushima et al., Cancer Res., 44: 5279 (1984), Shitar et al., Cancer Res., 47: 1267 (1987), Takada et al., Biochem. Biophys. Res. Commun., 179: 713 (1991 )).

Biosynthetic inhibitors for selectin ligand are such as a glycosyltransferase inhibitor which is used for biosynthesis of the ligands described above. Specifically, an inhibitor of sialyltransferase which transfers sialic acid to oligosaccharide receptor, and an inhibitor of fucosyltransferase which transfers fucose are included, (see, for example, sialyltransferase inhibitors disclosed in Japanese Patent Publication No. 247078/1993, and fucosyltransferase inhibitors disclosed in Wong et al., J. Am. Chem. Soc, 114: 7321 (1992)). Anti-selectin antibody means an immunoglobulin which recognizes selectin, selectively binds to selectin, and, as a result, inhibits intercellular adhesion. The antibody may be either a polyclonal antibody or a monoclonal antibody. The antibody can originate from any species and examples of the antibodies include mouse-, rat- or human-derived antibodies, chimera antibodies among human, rat and mouse antibodies, or humanized antibodies, etc.. Specifically, PB 1.3 disclosed in WO 9321956 is included. Selectins and fragments thereof mean membrane-binding selectins, soluble selectins, and peptides which are parts of selectin, etc.. Selectins and fragments thereof have an ability to inhibit cell adhesion, for example, adhesion of selectin to the foregoing ligands or derivative thereof which are immobilized on plastic wells. Peptides which are parts of selectin include ones disclosed in WO9404568.

An inhibitor for selectin-expression means a substance inhibiting the expression of selectin proteins. For example, dexamethasone is included (Yamamoto et al., 16th Annual Meeting, the Japanese Society of Inflammation (1995)).

A method for evaluating new selectin inhibitors using a pathologic animal having pleurisy can be used for determining their abilities of treating or preventing pleurisy. The method is also useful for studying effect of simulation on progress in human pleurisy.

The following working examples will illustrate that the pathologic animal of the present invention enables effective evaluation of substances including selectin inhibitors for their abilities of preventing or treating pleurisy. In the experiments, there were used a lipoteichoic acid derived from Staphylococcus aureus (Sigma) and, for selectin inhibitor, an anti-rat E- selectin monoclonal antibody, dexamethasone, and a sialyl Lewis X derivative.

Experiment 1 Animals and reagents used

Rat: male Wistar rat, 6-7 weeks old, Charles River Japan. Rats were anesthetized with ether and then administered with lipoteichoic acid at a dose of 0.1 mg/kg, at between 3rd and 5th ribs. After 4 hours, the animals were slaughtered by bleeding and then intrapleurally administered with 1 mL of PBS(-) containing phenol red, and infiltrate into pleural cavity was collected. Counting of cell numbers

The volume of infiltrate into pleural cavity was measured, washed with PBS(-) for several times, and then, the number of cells in the resultant suspension was counted by microcellcounter to calculate the total number of cells in the suspension.

Counting of the number of neutrophils

The number (cells/mL) of neutrophils in the infiltrate into pleural cavity was calculated as follows. Cell suspensions obtained above were subjected to Giemsa staining, and then the number of neutrophils was counted by microscope to calculate percentage of neutrophils in the cell suspension. Multiplying the total number of cells obtained above by the percentage of neutrophil provided neutrophil numbers in the infiltrate into pleural cavity. Calculating of survival rate of cells Survival rate of the cells was calculated after subjecting the cell suspension to trypan blue staining and then counting the number of unstained cells by microscope. The results demonstrated that lipoteichoic acid did not exhibit significant cytotoxicity. Statistical study Neutrophil numbers were represented by mean ±S.E.M.. The statistical significance was analyzed by Dannet's t-test or Student's t-test, and difference with the level of significance of 5% or less was determined as significant. Antibodies used

Anti-E-selectin monoclonal antibodies were prepared in conventional manner as follows. Rat E-selectin c-DNA was cloned using PCR by amplifying cDNA which was synthesized based on mRNA from rat lung stimulated with LPS. The rat E-selectin cDNA thus obtained was inserted into a vector, and the vector was used to transform mammalian cells, followed by cultivation of the cells. Rat E-selectin excreted in the supernatant of the cell culture was collected and purified. Immunization of mice was conducted using the purified E-selectin as an antigen. Hybridomas were prepared in conventional manner using lymphocytes obtained from the immunized mice, followed by screening of the hybridomas to select hybridomas producing an antibody which inhibits E-selectin mediated cellular adhesion. One of antibodies thus obtained was designated ARE-5 and used for further experiments. ARE-5 was intravenously administered at a dose of 0.5 mg/kg, 1.0 mg/kg, or 2.0 mg/kg one hour after administration of lipoteichoic acid. Results

The experimental results are shown in Figure 1. The administration of lipoteichoic acid into pleural cavity in rats induced neutrophil accumulation into the pleural cavity. An anti-E-selectin monoclonal antibody (ARE-5) significantly inhibited such neutrophil accumulation induced by lipoteichoic acid in a dose-dependent manner.

Experiment 2

Experiment 2 was conducted according to the procedure described in Experiment 1. Agent tested

Dexamethasone (Sigma) was suspended in 10% Nikkol(™) and the resulting suspension was intravenously administered at a dose of 0.5 mg of dexamethasone/kg (body weight), 2 minutes before administration of lipoteichoic acid. Results

The experimental results are shown in Figure 2. The administration of lipoteichoic acid to rats induced significant amount of neutrophil accumulation into the pleural cavity. Dexamethasone which is an inhibitor against expression of E-selectin significantly prevented such neutrophil accumulation induced by lipoteichoic acid.

Experiment 3

Experiment 3 was conducted according to the procedure of Experiment

1 , except for using mice (male ICR mice, 6-7 weeks old, Charles River Japan) instead of rats.

Agents used Dodecyl [(5-acetamido-3,5-dideoxy-α-D-glycero-D-galacto-2- nonulopyranosylonic acid)-(2→3)-0-(β-D-galactopyranosyl)-(1→4)-O-[α-L- fucopyranosyl-(1 →3)-0]-(2-deoxy-2-naphthamido-β-D-glucopyranoside)], a sialyl Lewis X derivative, was prepared by known-procedure (WO 9426760), dissolved in saline, and then the solution was intravenously administered at a dose of 30 mg of the derivative/kg (body weight), one hour after administration of lipoteichoic acid.

Results

The experimental results are shown in Figure 3. The administration of lipoteichoic acid to rats induced neutrophil accumulation into the pleural cavity. A sialyl Lewis derivative which is an inhibitor against neutrophil adhesion to E-selectin significantly prevented such neutrophil accumulation caused by lipoteichoic acid.

Pathologic animal having pleurisy of the present invention is useful for screening of medicines for preventing or treating inflammation such as pleurisy, for example, for screening chemical substances for the purpose of development of a new medicine.

Claims

1. A pathologic animal having pleurisy, which is prepared by administration of lipoteichoic acid.

2. The pathologic animal having pleurisy of Claim 1 , wherein the onset of pleurisy is mediated by a selectin.

3. The pathologic animal having pleurisy of Claim 2, wherein the selectin is E- selectin.

4. A method for preparing a pathologic animal having pleurisy characterized by administering lipoteichoic acid to an animal.

5. The method for preparing a pathologic animal having pleurisy of Claim 4, which method is characterized by the onset of pleurisy mediated by a selectin.

6. The method for preparing a pathologic animal having pleurisy of Claim 5, wherein the selectin is E-selectin.

7. A method for evaluating a medicine for preventing or treating inflammation, which comprises using a pathologic animal having pleurisy prepared by administration of lipoteichoic acid to an animal.

8. The method for evaluating a medicine of Claim 7, wherein the medicine is a selectin inhibitor.

9. The method for evaluating a medicine of Claim 8, wherein the selectin inhibitor is an E-selectin inhibitor.

10. A method for screening a medicine for preventing or treating inflammation, which comprises the steps of:

(a) administering a chemical substance to be tested to a pathologic animal having pleurisy prepared by administration of lipoteichoic acid to an animal; and

(b) determining ability of said substance for treating or preventing pleurisy in said pathologic animal.