AU613672B2

AU613672B2 - Treatment of diseases with paf-acether-antagonists and procedure for determining their efficacy

Info

Publication number: AU613672B2
Application number: AU23943/88A
Authority: AU
Inventors: Ruth Dr. med. Korth
Original assignee: Korth Ruth-Maria Dr
Current assignee: Korth Ruth-Maria Dr
Priority date: 1987-10-20
Filing date: 1988-10-17
Publication date: 1991-08-08
Anticipated expiration: 2008-10-17
Also published as: EP0312913B1; GR3004464T3; EP0312913A3; ES2032922T5; ES2032922T3; ATE72113T1; GR3018869T3; AU2394388A; JP2539257B2; JPH0291020A; DE3735525A1; EP0312913A2; DE3735525C2; EP0312913B2

Abstract

Substances which inhibit paf-acether binding sites on cells are used for the treatment of diseases caused by a reduction in the endothelial cell barrier.

Description

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AUSTRALIA

PATENTS ACT 1952 COMPLETE SPECIFICATION 61' 6

(ORIGINAL)

FOR OFFICE USE Application Number: Lodged: Complete Specification Lodged: Accepted: Published: o ~Priority: ,Related Art: 000 This document contains the amendments allowed under Section 83 by the Super- I vising Examiner on and is correct for prit:in- 0000 &0 0 TO BE COMPLETED BY APPLICANT O 04 o Name of Applicant: no 0 0 00 "Address of Applicant: °°oActual Inventor: 0 .0 o°"oAddress for Service: 0 0 co w co.rs.

o-a r Dr. med. Ruth Korth 32 rue des Carnets, F-92140 Clamart Dr. med. Ruth Korth ARTHUR S. CAVE CO.

Patent Trade Mark Attorneys Level 10 Barrack Street SYDNEY N.S.W. 2000

AUSTRALIA

0 0 0 CoComplete Specification for the invention entitled Treatment of Diseases with PAF-Acether-Antagonists and Procedure for Determining their Efficacy.

The following statement is a including the best method of full description of this invention performing it known to me:- 1 ASC 49

I

la Treatment of Diseases with PAF-Acether Antagonists and Procedure for Determining their Efficacy The invention refers to the treatment of diseases with pafacether antagonists and a procedure for determining their efficacy.

According to its chemical structure, paf-acether (platelet activating factor) is 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine Exp. Med., 136: 1356 1377, 1972; J. Biol.

Chem., 254: 9355 9358, 1979). It is known that paf-acether possesses powerful inflammatory and hypotensive properties, and that it induces coronary vasoconstriction and the acute formation of oedemas in the skin. Paf-acether receptors on platelets, polymorphonuclear neutrophiles and membrane preparations from human lung tissue have been described Immunol., 129: 1984, 1637 1641; Thromb. Res., 41: 1986, 699 706; Immunology 48: 141, 1983; Biochem. Biophys. Res. Commun. 128: 972, 1985).

The endothelium forms a cellular lining which protects the tissue. When the barrier of endothelium cells is broken, a variety of diseases occur. It is, for example, possible for Sfluid to escape from the tissue, which can lead to the formation i of oedemas. Bacterial and cerebral diseases, inflammation and allergies can also result from a lowering of the endothelium cell barrier. If the endothelium cell barrier is reduced, i spastic coronary constrictions can also be caused by an attack on the coronary vessels. Since the endothelium provides protection against parts of diseased tissue spreading, it combats the formation of metastases. Lesions o the endothelium likewise lead to an increased inflow of material, which causes reactions that can lead to arteriosclerosis.

Surprisingly enough, it has now been discovered that substances that inhibit the paf-acether binding sites on cells (pafacether antagonists) are suitable for treating diseases caused by the breakdown of the endothelium cell barriers.

ON7 S Sec. 77 r &1T J T 2 These substances are especially suitable for treating and preventing inflammations, allergies (including asthma), oedemas, bacterial diseases, vascular constrictions, especially in connection with the gastro-intestinal tract, the brain and the heart, such as spastic coronary constrictions, arteriosclerosis, arterial and venous thromboses, and anaphylactic and endotoxic shock. They are further suitable for reducing the tendency to form metastases. In addition, food ingredients appear to influence the paf-acether receptors on endothelium cells.

Furthermore, substances which inhibit the nidation of the fertilised ovum in early pregnancy (antinidation) can have an effect on the paf-acether receptors of the endothelium cells.

The substance that inhibits the paf-acether binding sites in this connection can be a hydrophilic triazolothieno-diazepine or an analogous substance. In addition, gingkolides and pafacether analogues, such as CV 3988, have proven suitable.

Triazolothieno-diazepines are described in Br. J. Pharmac.

(1987) 90: pages 139-146, gingkolides in "Blood and Vessel" (1985), No. 16: pages 558-572. Of the hydrophilic triazolothieno-diazepine compounds, WEB 2086 and 2098 are especially suitable. Of the gingkolides, BN 52020, BN 52021 and a mixture of BN 52020, BN 52021 and BN 52022, which is referred to as BN 52063, achieve the best results. Synthetic derivatives of the gingkolides can also be used.

Apart from its great efficacy, the gingkolide mixture BN 52063 stands out because it is relatively easy to manufacture, since it arises as a mixture in the course of processing the gingkolides, which means that it does not have to be broken down into individual components.

According to the invention, WEB 2086 and WEB 2098, CV 3988 and BN 52020, BN 52021 and BN 52063 in particular are used to prevent a penetration of the endothelium cell barrier by paf-acether and by cells stimulated by paf-acether.

ON K L _lb -3- The chemical formula of CV 3988 is rac-3-(N-n-octadecyl carbamoyl-oxy)-2-methoxypropyl 2-thiazolioethyl phosphate; the formula of WEB 2086 is 3-(4-(2-chlorophenyl)- 9-methyl-6H-thieno(3,2,-f) triazolo-(4,3-a)diazepine-2yl)-l-(4-morpholinyl)-1-propanon; for WEB 2098 it is 3-(4-(2-chlorophenyl)-9-cyclopropyl-6Hthieno (1,2,4)triazolo-(4,3-a) (1,4)diazepine- 2yl-l-(4-morpholinyl)-l-propanon; for BN 52020 9H-1, 7a- (epoxymethano) -lH, 6aH-cvclopenta[c]furo[2,3-b] furocyclopenta [1,2-d]furan-5,9,12-(4H)-trion, 3-tert-butylhexahydro-4, 4b-11-trihydroxy-8-methyl; for BN 52021 9H-1, 7a-(epoxymethano)-1H,6aH-cyclopenta [c] furo furo-[3',2':3,4] cyclopenta furanoa 0o 5,9,12(4H)-trion, 3-tert-butylhexahydro-4, 7b-dihydroxy- 8-methyl; and the formula for BN 52022 is 9H-1,7a- S(epoxymethano)-lH, 6aH-cyclopenta furo 00 a 0B o cyclopenta furan-5,9,12(4H)-trion, 3-tert-butylhexahydro-2,4,7b,ll-tetrahydroxy-8-methyl.

As has now been discovered, the endothelium cells exhibit binding sites or receptors for paf-acether. By 000 interaction with these receptors, mediators are formed, o Coldngpf o such as histamine, prostaglandins, etc., including pafacether itself, which attack cr destroy the endothelium 0o 04 cells.

b It is also possible that paf-acether bound to the receptors of endothelium cells leads to the adherence of cells such as neutrophiles, eosinophiles and the like, which cause the formation of mediators.

Thrombocytes, neutrophiles and eosinophilcs for their part have receptors for paf-acether. There is probably a further mechanism that can lead to the penetration of the endothelium cell barrier, consisting in the fact that the binding of paf-acether to the receptors of these cells can lead to their adhering to endotheliumi cells or to the production of mediators as already mentioned, which then attack the endothelium cells.

iI: t' o 0 In order to counteract this, according to the invention, substances are used which on the one hand directly inhibit the binding of paf-acether to the endothelium cells, and on the other hand also substances which block the binding of paf-acether to other cells, such as thrombocytes, neutrophiles and eosinophiles, and thus prevent these mediators from attacking the erdothelium cells indirectly.

In other words, according to the invention, substances are used which inhibit the binding sites of paf-acether to endothelium cells; because of the formation of mediators if paf-acether is bound to other cells, and also because of the impairment of the endothelium cells by o e these mediators, it is, however, possible to use compounds which inhibit the binding sites of paf-acether to these cells.

oj The substances which inhibit the binding sites of pafacether can be administered by injection, for example, Sbut also orally, percutaneously and by inhalation.

In order to conduct a quick and simple test on the effect of the substances with regard to their antagonistic °oa0o activity vis-a-vis paf-acether receptors, i.e. to use a screening procedure, for example, to find effective antagonists to paf-acether receptors, which can then be taken into consideration for treating the diseases mentioned, then according to the invention, the best method is to proceed as follows: a) endothelium cells are cultivated, b) the endothelium cells are washed, c) a given quantity of endothelium cells is mixed with a given quantity of marked paf-acether and of the antagonist to be determined, d) a given quantity of endothelium cells is mixed with a given quantity of marked paf-acether, 9 0289g/MS e) the endothelium cells are and d) in each case, separated from the mixtures c) 000 090 0 0 0 a 0 4a 5 f) the quantity of marked paf-acether bound to the endothelium cells is measured in each case, and g) the efficacy of the paf-acether antagonist is determined from the relationship between on the one hand the quantity of marked paf-acether which is bound to the endothelium cells according to c) in the presence of the antagonist, and on the other hand the quantity of marked paf-acether which is bound to the endothelium cells according to d) in the absence of the antagonist, related to the same number of endothelium cells.

o. The cultivation of the endothelium cells in accordance with step a) according to the invention is preferably to be performed in a culture medium containing serum, and in particular calf serum. A culture medium-containing no So serum can, however, also be used.

In this context, the preferred method is to cultivate confluent endothelium cells in the culture medium, which oo generally grow on the floor of the culture vessel.

In order to remove serum, and with it enzymes that break ,o down paf-acether, such as acetyl hydrolase, the endothelium cells are then washed in accordance with step b) of the procedure according to the invention. For this pur- S* pose, a buffered isotonic washing liquid is used, which should preferably contain delipidated serum albumin, such as human serum albumin (HSA) or bovine serum albumin (BSA), including endoxin-free serum albumin.

Then, in accordance with steps c) and d) according to the invention, a binding study is carried out on the endothelium cells with marked paf-acether and the antagonist to be determined on the one hand, and marked pafacether without the antagonist on the other hand. It is preferable to choose radioac'-ively marked paf-acether, such as tritium-marked paf-acether, for use as the marked paf-acether.

I

I O 6 It is preferable to conduct the binding studies at a room temperature of less than 20"C, and preferably at 2- 6°C, so that the enzymes contained in the endothelium cells, such as phospholipases or acetyl hydrolases, do not lead to a breakdown of the paf-acether. The preferred incubation period is 10 to 30 hours.

Since, as a phospholipide, paf-acether does not dissolve in water, it is furthermore preferable to conduct the binding studies in accordance with steps c) and d) i' the. presence of delipidated serum albumin, such as HSA or BSA, including endoxin-free serum albumin, and in the process, the preferred method is to add calcium and 0 04 S° magnesium ions.

The task of the serum albumin in this context is to bind c.Oo the paf-acether and antagonist which is bound unspecifio° cally to the endothelium cells, i.e. not to the recepo tors, or in other words to remove it from the endothelium cells.

o C 0 o o Between steps e) )nd the confluent endothelium cells are detached from their base (culture dish) and from one ooOs another.

For this purpose, it is preferable to carry out some additional steps in order to obtain endothelium cells which essentially exhibit the greatest possible amount of paf-acether or antagonists bound specifically to the receptors. For this purpose, the endothelium cells are first preferably mixed with an isotonic liquid, i.e. in particular a physiological saline solution, so that in the process, calcium and magnesium ions and unspecifically bound paf-acether and antagonists are sepaLated from the endothelium cells.

f- I" _r 0 7 The preferable method is then to have a second washing step, in which an isotonic liquid cooled to 5°C or less, preferably to about 0°C, is added to the enduthelium cells; it is preferable for this liquid to contain EDTA.

Because of the low temperature and the EDTA, which binds the calcium ions in the endothelium cells, the confluent endothelium cells contract, so that in the process, they are detached from the culture vessel or other base and also from one another, i.e. the confluent layer of endothelium cells is destroyed. At the same time, the low temperature of the washing liquid means that unspecifically bound paf-acether and antagonists are more easily detached from endothelium cells.

In order to separate the endothelium cells treated in this way from the liquid medium, it is preferable to filter them. They are very easy to filter, since in the detachment method described the endothelium cells are not damaged, let alone destroyed. At the same time, filtering constitutes a relatively precise and therefore very effective procedure for separating from the endothelium cells the remaining marked paf-acether which is unspecifically bound.

After this, the quantity of marked paf-acether which is (specifically) bound to the endothelium cells is determined; if radioactively marked paf-acether is used, only the radioactivity of the endothelium cells bound in the filter is measured. The radioactivity bound in the filter where there are no endothelium cells is subtracted from these values.

By drawing calibration graphs, which are obtained with varying quantities of the antagonist in accordance with step it is thus possible to obtain the efficacy of the antagonist as a 50 inhibitory value, i.e. as that quantity of the antagonist which, in relation to a given quantity of endothelium cells, leads to a 50 inhibition of the reversible paf-acether binding.

*~I

II~"LX-~ -8 The procedure according to the invention was tested successfully with the following paf-acether receptor antagonists: hydrophilic triazolothieno-diazepine, WEB 2086 and 2098, the gingkolides BN 52020, BN 52021 and a mixture of BN 52020, BN 52021 and BN 52022, and with CV 3988.

Monoclonal antibodies directed against these receptors can also be taken into consideration as paf-acether receptor antagonists according to the invention. The antibodies can also be used in a marked or fluorescent form.

In this case, before the binding study in accordance with steps c) and a pre-incubation of the endothe- S' lium cells is carried out with surplus antibodies. The binding studies in accordance with steps c) and d) can also be carried out with radioactively marked or fluoro escent paf-acether antagonists or analogues, which are S then displaced from the binding by non-marked paf- .o acether. In the same way, in a process according to the invention, it is possible for the paf-acether to be S"o0 replaced by paf-acether analogues, and for the pafacether antagonist to be replaced by analogues of such o antagonists; in other words, in steps c) f) and g) o0 of the procedure in accordance with Claim 4, the marked and unmarked paf-acether can be replaced by marked or unmarked paf-acether analogues, paf-acether antagonists or analogues of these antagonists.

I The procedure according to the invention is suitable not i only for determining antagonists of the receptors for paf-acether as such, but also for determining paf-acether complexes, especially the paf-acether lipoprotein complex, which is referred to as "PEAK and which is described in Fed. Proc. 46, 1987 page 1468.

The following example serves to explain the invention more clearly.

L I 9 1. Production of the endothelium cells.

Human endothelium cells were isolated from the umbilical vein Clin. Invest., 1973, 52: 2745-2756). In other words, a cannula was inserted into the vein and filled with a 0,1 collagenase solution (type I, Sigma, St. Louis, MO, USA).

After a 10-minute incubation period at 370C, the detached cells were rinsed out and collected by means of centrifugation. The cells were re-suspended in a Hams-F-12 medium with thermo-inactivated foetal calf serum (FCS Hyclone, Logan, Utah, USA), 1 ultroser SF (IBF, Villeneuve la Garenne, France) and 90 microgrammes/ml of heparin, with the addition of 50 units/ml of penicillin and 50 microgrammes/ml of streptomycin (Science 1983, 222: 623-625). The cells were incubated in a 25 sq. cm. culture flask (Primaria, Falcon, Labware, Oxnard, California, USA) for one hour. The nonadhering cells were then washed away carefully, and the adhering cells were cultivated further in fresh culture medium, with the medium being changed every 2 days. When the cultures had reached confluence (3 to 5 days), the cells were harvested by being briefly exposed to trypsin EDTA (Gibco, Paisley, Scotland) and put in a 35 mm culture dish (Primaria, Falcon) in a 1 3 to 1 5 split relation. The cells were then allowed to continue growing in a Hams-F-12 medium with 15 FCS, 1 ultroser SF and 90 microgrammes/ ml of heparin, with the medium being changed every two to three days. The cells reached confluence after 4 to 6 days, with the number of cells per dish amounting to 5,2 0,15 x 105. The cells from the first process were used throughout the whole investigation. The cultures were determined to be endotheliur cells on the basis of morphological criteria and by means of indirect immunofluorescence, using a specific anti-serum for human factor VIII antigen (Nordic Immunol., Tilburg, Netherlands), which is a standa-rd marking substance for endothelial cells. The endothelial cell cultures did not contain any monocytes/macrophagecontaining cells on the basis of morphological criteria and by means of indirect immunofluorescence, using monoclonar antibodies for alpha 2 -macroglobulin.

0 Sec.

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ti t' J 10 2. Paf-acether binding to endothelial cells.

Of the confluent human endothelial cells, i.e. those which had grown together, the culture medium was twice carefully washed off with Tyrode's buffer containing 0.25 bovine serum albumin (BSA). To the confluent endothelial cells the following were added at varying time intervals 5, 15, 30 min.): 900 microlitres of Tyrode's buffer (pH 0.25 BSA (Sigma), 1.3 mM Ca 2 100 microlitres of H-pafacether (0.065 nM) (l-0- 3 H-octadecyl-2-acetyl-snglyceryl-3-phosphorylcholine 80 Ci/mM, Amersham, Bucks., England) with and without 500 nM unmarked 0, paf-acether (l-0-octadecyl-2-acetyl-sn-glyceryl-le phosphorylcholine) (Bachem, Bubendorf, Switzerland).

0o 0 a oa In a second experiment different concentrations of o 3 H-paf-acether (0.0325 0.65 nM) were incubated for minutes in the presence or absence of unmarked o paf-acether or its enantiomers (3-0-hexadecyl-2- 0 o acetyl-glyceryl-l-phosphorylchol ine) (500 nM) as o 00 o0 9 described above.

.0 .0 ooo* In further tests, the paf-acether antagonists

BN

52021 (60, 10, 6 micro-M) (IHB-IPSEN, Le Plesses- Robinson, France), CV 3988 (30, 10, 3 micro-M) (Takeda Chemical Ind., Osaka, Japan) or vehicles (dimethyl sulphoxide or an isotonic NaCl solution) were added to confluent endothelium cells together with 0.65 nM3H-paf-acether.

After 30 minutes, the free activity was washed off the confluent endothelium cells twice with Tyrode's buffer (pH 6.4) containing BSA and then once with cold isotonic NaC1 solution. The cells were separated by adding cold isotonic EDTA NaCI solution (5 mM), after which they were incubated for 30 60 minutes Sat 0 4"C. The detached cells were separated from the medium by vacuum filtration with a millipore vacuum system (Molsheim, Federal Republic of Germany).

,L 1 (AV P 11 The incubation buffer was likewise filtered in order to avoid a loss of cells during the washing process after the binding attempt. The filters were washed twice with 10 ml of cold Tyrode's buffer, and the radioactivity bound to the filters was determined under standard conditions in PCS (Amersham). The radioactivity bound to the filters in the absence of endothelium cells was subtracted from the radioactivity in the presence of endothelium cells. The bound radioactivity was calculated in fmol H-paf-acether bound to 5.20 0.15 x 105 endothelium cells.

After an incubation period of 30 minutes with growing concentrations of paf-acether antagonists, the cells were released by meins of a cold EDTA NaCl solution and then separated by vacuum filtration. The 3 H-paf- 4 acether binding was calculated in fmol bound to 5.2 0.15 x 105 endothelium cells.

The following table contains the results of the spe- Scific 3 H-paf-acether binding in fmol to 5.2 0.15 x confluent endothelium cells.

STable 1 CV 3988 BN 52021 fmol fmol 3 micro-M 2.26 1.4 6 micro-M 1.6 1.4 micro-M 2.70 1.9 10 micro-M 4.8 micro-M 4.40 1.4 60 micro-M 3.9 3.1 I.e. when 3 micro-M of CV 3988 are added, the pafacether binding to 5.2 105 endothelium cells declines by 2.26 1.4 fmol, and by 1.6 1.4 fmol when 6 micro-M of BN 52021 are add&d, etc. In other dords, CV 3988 constitutes a more effective antagonist than BN 52021.

12 Instead of an incubation period of 30 minutes at it is also possible to incubate for 20 hours at 4°C, though in this case it is preferable for the antagonists to be pre-incubated for 15 minutes. Among other things, this prevents a metabolisation of the ligands.

4. The paf-acether binding to endothelium cells in accordance with the above experiment under No. 2 was repeated, with the exception that instead of BN 52021 or CV 3988, hydrophilic triazolodieno-diazepine, WEB 2086 10, 100, 1 000 nM) was used.

o o The following table 2 contains the results.

T a b l e 2 WEB 2086 1 nM 0.J6 0.2 fmol nM 1.5 1.1 fmol 1 100 nM 2 1.1 fmol 1 000 nM 3 0.7 fmol SI.e. when 1 nM of WEB 2086 is added, the paf-acether 0\ binding to 5.2 x 105 endothelium cells declines by 0.66 0.2, and by 3 0.7 fmol when -1 7 nM are o 'added.

O

Claims

1. A method of treating a disease caused by a lowering of the endothelium cell barrier comprising administering to a subject requiring said treatment an effective amount of at least one paf-acether antagonist.

2. The method according to claim 1, wherein the paf-acether antagonist is a hydrophilic triazolothieno-diazepine or an analogue thereof, a ginkgolide or a synthetic ginkgolide derivative, or an analogue of the paf-acether.

3. The method according to claim 2, wherein the hydrophilic triazolothieno-diazepine is selected from WEB 2086 or WEB 2098 (as herein defined).

4. The method according to claim 2, wherein the ginkgolide is selected from BN 52020, BN 52021, or is a mixture of BN 52020, BN 52021 and BN 52022 (as herein defined).

5. The method according to claim 2, wherein the paf-acether analogue is CV 3988 (as herein defined).

6. A procedure for determining the efficacy of a paf-acether antagonist, characterised by the following steps: a) endothelium cells are cultivated, b) the endothelium cells are washed, c) a given quantity of endothelium cells is mixed with a given quantity of marked paf-acether and of the antagonist to be determined, d) a given quantity of endothelium cells is mixed with a given quantity of marked paf-acether, e) the endothelium cells are separated from the mixtures c) and d) in each case, ,o oi 0,0 0 .0 0 o 0 0 0 0 .0 00 0< 01 00 coool 0 0< 0 00 04 00 0001 0000 <o~<0 0 00 13 0289g/MS f) the quantity of marked paf-acether bound to the endothlium cells is measured in each case, and g) the efficacy of the paf-acether antagonist is determined from the relationship between the quantity of marked paf-acether which is bound to the endothelium cells according to c) in the presence of the antagonist on the one hand, and the quantity of marked paf-acether which is bound to the endothelium cells according to d) in the absence of the antagonist on the other hand, with reference to the same number of endothelium cells.

7. The procedure according to claim 6, characterised in that in step a) confluent endothelium cells are cultivated, which are detached after the separation of the endothelium cells according to e). o 0 0 8. The procedure according to claim 7, characterised in that S the endothelium cells are detached in an isotonic liquid cooled to 5 C or lower.

9. The procedure according to claim 8, characterised in that the isotonic liquid contains EDTA. The procedure according to any one of claims 6 to 9, characterised in that serum albumin is added to the medium containing endothelium cells, marked paf-acether and antagonists in accordance with c) and to the medium containing endothelium cells and marked paf-acether in accordance with d) in each case.

11. The procedure according to any one of claims 6 to characterised in that when the endothelium cells are washed in i I 14 0289g/MS accordance with this is done with an isotonic liquid containing serum albumin.

12. The procedure according to any one of claims 6 to 11, characterised in that the separation in accordance with e) is carried out by means of filtering.

13. A method of treating a disease caused by lowering of the endothelium barrier, substantially as herein described with reference to any one of the examples thereof. DATED this 26th day of April, 1990. DR. MED. RUTH KORTH By Her Patent Attorneys ARTHUR S. CAVE CO. o 0 0000 0OC 000 000 O 1oa1 15