FR2778851A1 - Composition for retarding senescence, autodestruction and hemolysis of erythrocytes in vitro or in vivo - Google Patents

Abstract

Composition for retarding senescence, autodestruction and hemolysis of erythrocytes comprises specific inhibitors of the active site of cysteine proteases.- ACTIVITY - None given.- MECHANISM OF ACTION - Cysteine protease inhibitor

Description

The invention relates to a composition for delaying senescence,

  self-destruction and hemolysis of

  erythrocytes, which includes cysteine inhibitors-

  proteases, particularly inhibitors of caspases and calpains. These inhibitors are preferably chosen from analogues of triou tetrapeptides

  cysteine recognition and cleavage site-

  proteases. They will be used alone or as a mixture, in particular in the conservation environments of

  erythrocytes for blood transfusions.

  It is known that erythrocytes have a lifespan of 120 days in the bloodstream. Then they are phagocytosed by macrophages. This phagocytosis is preceded and caused by numerous physiological and morphological disturbances within erythrocytes, in particular the reduction in their size, the appearance of pseudopods, the emission of vesicles, the desialylation of the membrane glycoconjugates and the outsourcing of phosphatidylserine ( going from the inner layer to the

  outer layer of the lipid layers of the membrane).

  It has been shown that these last two events, desialylation and the appearance of phosphatidylserine, which are considered as markers of senescence, are directly associated with the mechanism of capture by macrophages (Bratosin et al. Glycoconjugate J. 12, 1995, 258-267; Bratosin et al. Comptes Rendus Acad. Sci. 320,

1997, 811-818).

  It is also known that, after blood transfusion,% of transfused erythrocytes disappear from the circulation after 24 hours and 70% after 3 days. This phenomenon has also been attributed to the senescence of erythrocytes during their storage and to their phagocytosis

after transfusion.

  In addition, self-destruction and hemolysis of erythrocytes already begins in the blood bags for

their storage.

  Any improvement in the storage conditions of erythrocytes intended for transfusions, making it possible to preserve their integrity and their physiological activity, would therefore constitute considerable progress in the transfusion field. It is an object of the present invention to provide such an improvement. Although erythrocytes are cells without nucleus and without mitochondria, the Applicants have hypothesized that the phenomena associated with erythrocyte senescence and self-destruction could be assimilated to a mechanism of apoptosis, which has never been considered previously. On the contrary, it has also been published recently that "erythrocytes cannot undergo apoptosis since they lack a nucleus" (Boas et al. Proc. Natl. Acad. Sci. 95-1998-3077). The Applicants have therefore studied the effect of known inhibitors of apoptosis on markers of

erythrocyte senescence.

  Among various families of known inhibitors, the Applicants have chosen oligopeptides, mainly tri- and tetrapeptides and analogs or derivatives thereof which have already been described as inhibitors of caspases and calpains. These two families of enzymes are proteases which participate in a cascade of reactions

  associated with cytoplasmic apoptosis.

  - Numerous studies have highlighted the role of various families of intracellular proteases, in particular cysteine proteases, in modulating the activity of other cellular proteins and in balances between activators and inhibitors of various cellular functions (see reviewed by Sorimachi et al.

Biochem. J. 328, 1997, 721-732).

  Among these proteases, the caspases specifically cleave after an aspartic acid residue (see review by Cohen, Biochem. J. 326, 1997, 1-16). So far, ten caspases have been identified that participate in a cascade of nucleus and cystoskeletal protein cleavage reactions that characterize the phase

effector of apoptosis.

  On the other hand, the calpains combine a domain with cysteine protease activity and a domain which binds calcium; they thus participate in various cellular functions. Besides their well-described role in brain and muscle dysfunctions, calpains also seem to participate in the process.

  apoptosis. (Sorimachi - same reference).

  - The study of the mode of action of caspases has involved the design of synthetic inhibitors which are oligopeptides whose sequence is analogous to the tri- or tetrapeptide sequence of the recognition and

  cleavage present on the natural substrates of the caspases.

  Thus the DEVD tetrapeptide (Asp-Glu-Val-Asp) corresponds to the cleavage site of the poly-ADP-ribose polymerase and YVAD (Tyr-Val-Ala-Asp) corresponds to that of

ICE (IL-11-converting enzyme).

  YVAD inhibits caspases 1 and 4; DEVD inhibits caspases 3 and 7. Various derivatives (aldehyde, nitriles, chloromethyl, fluoromethyl, methyl ketones, etc.) have also been designed (see reviews: Nicholson and Thornberry; TIBS 22, 1997, 299-306; Villa and al.

TIBS 22, 1997, 388-393).

  A modified tripeptide, Z-VAD k (benzyloxycarbonyl-

  Val-Ala-Asp-fluoromethyl ketone) exerts an inhibition

irreversible on caspases.

  Therapeutic applications of these inhibitors have already been envisaged for the treatment of pathological conditions associated with apoptosis phenomena, such as fulminant hepatitis, myocardial infarction and

ischemic strokes.

  - Inhibitors of the cysteine protease function of calpain have also been described (Sorimachi - same

  reference): the molecule known under the code E-64 (trans-

  epoxysuccinyl-4-leucylamido- (4-guanidino) -butane), isolated from a culture of Aspergillus japonicus, as an inhibitor of papain, as well as two derivatives

  E-64c (N- [N (L-3-trans-carboxyoxirane-2-carbonyl) -L-

  leucyl] isoamylamine) and E-64d (N- [N- (L-3-trans-

  ethoxycarbonyloxyoxirane-2-carbonyl) -L-leucyl] iso-

  amylamine) and leupeptin (N-acetyl-Leu-Leu-argininal) isolated from different strains of Actinomycetes, as well as close analogues or synthetic derivatives such as

  N-acetyl-Leu-Leu-norleucinal, N-acetyl-Leu-Leu-methio-

  ninal, benzyloxycarbonyl-Leu-Leu-leucinal, etc ...

  These molecules inhibit cysteine proteases to varying degrees; some have already been used for the treatment of muscular dystrophies linked to these proteases. These inhibitors are marketed (in particular by the companies SIGMA and BACHEM) and are offered in the form

  apoptosis-inhibiting reagents.

  The Applicants have used these various caspase and calpain inhibitors and have been able to show that, surprisingly, they also had an inhibitory effect on four well-defined markers of erythrocyte senescence: - morphological changes, the externalization of phosphatidylserine - erythrophagocytosis

- hemolysis.

  These observations therefore support the initial hypothesis and make it possible to assimilate senescence and self-destruction of erythrocytes to a phenomenon of apoptosis. The Applicants have deduced therefrom practical applications, in particular for improving the conditions of

  conservation of erythrocytes intended for transfusions.

  Thus, the invention relates to a composition for delaying senescence, self-destruction and hemolysis of erythrocytes, which comprises inhibitors specific for the active site of cysteine proteases. Said inhibitors preferably have a structural analogy with

  peptide motif specifically recognized by cysteines-

  proteases. More particularly, a first group of inhibitors presents a structural analogy with a tetrapeptide motif specifically recognized by caspases. A second group of inhibitors presents a structural analogy with a tripeptide motif specifically recognized by calpains. The composition according to the invention preferably comprises a mixture of inhibitors of the analogous type of the tetrapeptide motif and

  inhibitors of the tripeptide motif analogous type.

  The inhibitors of the first type are chosen from

  preferably among the Asp-Glu-Val-Asp, Tyr- tetrapeptides

  Val-Ala-Asp or the tripeptide Z-Val-Ala-Asp or derivatives thereof modified upstream or downstream of the aforementioned specific sequence, such as those which have been described in the literature (references cited above) ; they

  can be used alone or as a mixture.

  The inhibitors of the second type are preferably chosen from leupeptin or its derivatives and the molecule E-64 or its derivatives, such as those which have been described in the literature (references cited above);

  they can be used alone or as a mixture.

  Other molecules could also be synthesized or used as inhibitors of the same caspases and calpains, which act by any mechanism on the active site of these same enzymes. These molecules could also be used as inhibitors in the composition according to the present

  invention, as well as structural analogs.

  The invention also relates to the erythrocyte preservation method which makes it possible to delay senescence, self-destruction and hemolysis, by adding the composition of inhibitors in the conservation medium, to a final concentration of inhibitors. between 0.1 and 100 mM, for a final hematocrit

close to 30%.

  The composition according to the invention also finds uses in the preparation of medicaments intended for the treatment of various pathologies linked to early or aberrant manifestations of senescence or of self-destruction and of hemolysis of erythrocytes and associated, in particular, with various forms of anemia. The following examples illustrate the invention without

however limit its scope.

  Figure 1 is a scanning electron microscope photograph which illustrates the effect of

  inhibitors on erythrocyte morphology.

  FIG. 2 is a photograph with a scanning electron microscope which illustrates the effect of the inhibitors on the capture of erythrocytes by macrophages. EXAMPLE I I-A-Induction of erythrocyte self-destruction in vitro A erythrocyte population always includes a certain percentage of senescent or self-destructive cells, the morphological and physiological characteristics of which have been described more

high.

  We can speed up the process of senescence and induce self-destruction of erythrocytes, in vitro, by keeping them for 24 to 48 hours at 37 C. (They are normally stored at 4 C for 2 to 6 weeks in

transfusion centers).

  One can also artificially trigger the process of self-destruction by the action of a specific ionophore of the calcium channels, known under the number of

  code A-23187 (marketed by Calbiochem-Novabiochem).

  This ionophore increases the solubility of calcium ions in lipid media and thus facilitates their

  penetration through cell membranes.

  The following senescence markers have been chosen for subsequent studies because they are easy to observe and / or quantify: 1Alteration of the discoid appearance and appearance of pseudopodia (erythrocytes at the "echinocyte" stage), then filiform pseudopodia (erythrocytes at the "spheroechinocyte" stage). 2- Capture and phagocytosis of erythrocytes by macrophages.

  3- Outsourcing of phosphatidylserine.

4- Hemolysis of erythrocytes.

  The morphological aspect of erythrocytes is analyzed

  in scanning electron microscopy.

  The phagocytosis of erythrocytes by macrophages is measured either by observation and direct counting on view under a microscope, or after labeling with fluorescent dye PKH-26 by flow cytofluorimetry, as described by Bratosin et al. (Cytometry 30, 1997, 269). Peritoneal macrophages from mice (females of

  Swiss breed, 6 to 8 weeks old).

  The outsourcing of phosphatidylserine is measured by labeling with annexin V conjugated to fluorescein (annexin V-FITC) and flow cytofluorimetry, as described by Kuypers et al. (Blood 87, 1996,

1179).

  Hemolysis is assessed by hemoglobin assay

  released into the conservation environment.

  In the results tables, the inhibitor peptides will be designated by the letter code:

DEVD, YVAD, Z-VAD.

  I-B- Conditions for studying inhibitors The erythrocytes are suspended in HEPES buffer supplemented with 2.5 mM CaC12, at a concentration of

107 cells per ml.

  - The erythrocytes are maintained at 37 C for 48 hours - or incubated in the presence of ionophore A-23187 5 M at 37 C for 3 hours

  - Asp-Glu-Val-Asp (DEVD), Tyr-Val- inhibitors

  Ala-Asp (YVAD), leupeptin and E-64 are added to the

  suspension of erythrocytes at a concentration of 200 gM.

  The mixture is maintained at 20 ° C. for 30 minutes. The Z-

  Val-Ala-Asp (Z-VAD) is used at 40 LM.

  IC- Evaluation of the effect of the inhibitors on the morDholoqie of the ervthrocvtes Fiaure 1 shows the aspect of the erythrocytes observed in scanning electron microscopy: A- native erythrocytes Berythrocytes treated with the ionophore A-23187 C- erythrocytes brought into contact with DEVD inhibitor before ionophore treatment The figure is presented by way of example. The same results are obtained with erythrocytes maintained at 37 ° C. for 48 hours (B) and with the YVAD inhibitors,

E 64, and leupeptin (C).

  I1 clearly appears in FIG. 1C that almost all of the treated erythrocytes have retained their native discoid appearance, while in FIG. 1B a proportion

  important erythrocytes are spheroechinocytes.

  The latter are the only ones to outsource phosphatidylserine and to be phagocytosed (Bratosin et al.

  Reports of the Acad. Sci. Paris, 320, 1998, 811).

  ID- Evaluation of the effect of the inhibitors on the capture and on the Dhaaocvtose of the ervthrocvtes by the macrophaaes With the same conditions of incubation and inhibitory treatment, one evaluated, by counting on images of electron microscopy, the number of erythrocytes captured by mouse macrophages in

  culture, after contacting for 2 hours.

  The counting is carried out on 500 macrophages per sample. The results are presented in Table I and illustrated in FIG. 2. They show a significant reduction in capture after treatment with the inhibitors. Table I Treatment of erythrocytes Number of erythrocytes captured by 100 macrophages none 6 ionophore A 23 187 500 A 23 187 + inhibitors:

DEVD 70

YVAD 15

leupeptin 25

E-64 30

  DEVD + leupeptin 14 Fiaure 2 shows the capture of human erythrocytes by mouse peritoneal macrophages, observed by scanning electron microscopy: A- native erythrocytes B- erythrocytes treated with ionophore A-23187 C- erythrocytes brought into contact with leupeptin

before treatment with ionophore.

  The figure is presented as an example.

  The same results are obtained with erythrocytes maintained at 37 ° C. for 48 hours (B) and

with other inhibitors (C).

  On the other hand erythrophagocytosis by macrophages was measured by cytofluorimetry after

labeling with PKH-26.

  The results are presented in Table II, expressed as a percentage of inhibition of erythrophagocytosis. The measurements are carried out on 5,000 cells for

each condition.

Table II

  Percentage inhibition of erythrophagocytosis Inhibitors: at 37 C after treatment with ionophore

A-23187

DEVD 50 + 8 60 + 10

YVAD 56 + 12 40 + 8

Z-VAD - 41 + 12

Leupeptin 82 + 12 49 + 13

E-64 80 + 19 37 + 8

DEVD + E-64 92 + 3 95 + 3

  I-E- Evaluation of the Effect of Inhibitors on the Outsourcing of Phosphatidylserine With the same conditions of incubation and inhibitory treatment, the capacity of

  erythrocytes to fix annexin V-FITC.

  After washing in isotonic phosphate buffer, the erythrocytes (105 cells in 100 g1) are incubated in the presence of 0.1 gg of annexin VFITC, for 15 min at

 C, in the dark.

  The percentage of fluorescent cells (annexin-

  positive) is determined by flow cytofluorimetry (on FACScan cytofluorimeter-Becton-Dickinson, with LYSIS program), by counting from 5 to 10,000 cells per sample. The results are presented in the table

following.

Table III

  Percentage inhibition of phosphatidylserine outsourcing Inhibitors at 37 C after treatment with ionophore

A-23187

DEVD 83 + 12 90 + 6

YVAD 82 + 4 82 + 10

Z-VAD - 71 + 10

Leupeptin. 89 + 8 83 + 13

E-64 82 + 8 96 + 4

DEVD + E-64 96 2 94 + 2

  Note: Z-VAD being added with ethanol or DMSO causes the lysis of erythrocytes during a prolonged incubation. The 37 C test is therefore not performed. I-F- Evaluation of the Effect of Inhibitors on Self-Destruction of Erythrocytes by Hemolysis Human erythrocytes were incubated in the presence of ionophore A-23187 at 37 C for 8 and 20 hours, in

  absence and presence of inhibitors.

  Hemolysis is measured and expressed in mg

  hemoglobin released per liter of medium.

  The results presented in Table IV show

  that the inhibitors protect the erythrocytes from lysis.

Table IV

  Hemoglobin in the storage medium (mg / 1) Treatment of erythrocyte incubation times 8 hr 20 hr - ionophore in the absence of inhibitor 60 195 - ionophore in the presence of inhibitors:

DEVD 7 46

YVAD 8 41

  leupeptin 9 27 DEVD + leupeptin 8 32 YVAD + leupeptine 9 24 ExemDile II In vivo clearance of mouse cells To assess the effect of inhibitors on erythrocytes in storage conditions, then transfused,

  we have developed a Balb-c mouse study model.

  Mouse erythrocytes are isolated by

  centrifugation (2,000 g, 5 min at 4 C) of heparinized blood.

  After 3 washes, in phosphate buffer at pH 7.2 they are taken up in HEPES buffer supplemented with 0.1% of

  human serum albumin. The final hematocrit is 10%.

  The erythrocytes are incubated for 4 days at 20 C (instead of 37 C for human erythrocytes due

  of the greater fragility of mouse erythrocytes).

  Then, the erythrocytes are centrifuged and labeled with fluorescent dye PKH-26 (under the same conditions used for human erythrocytes). We take 100 g of erythrocyte pellet in 100 μl of

  phosphate buffer to inject them into the retro sinus

mouse orbital.

  Blood samples are then taken over time to measure presence, then clearance.

injected erythrocytes.

  The measurement is carried out by flow cytofluorimetry. Each measurement relates to 10,000 cells. The first blood test is carried out 1 hour after the injection, so that all

  injected erythrocytes have passed into circulation.

  The same procedure is applied to erythrocytes incubated at 20 ° C. in the presence of inhibitors to

a concentration of 2 mM.

  Freshly collected erythrocytes are used as a control. The results presented in Table V show that the clearance of untreated erythrocytes is finished from the 6th hour while the erythrocytes treated with

  inhibitors are still present after 48 hours.

Table V

  Number of erythrocytes Treatment of the marks remaining in the circulating erythrocytes (out of 10,000 counted) after 1 hr 6 hrs 20 hrs 48 hrs none 364 343 317 305 4 days at 20 C 34 11 11i 10 4 days at 20 C in the presence of 2 mM of

* DEVD 276 260 220 210

  * YVAD cmk 359 340 330 300 * leupeptine 219 230 250 230 * YVAD cmk + leupeptine 422 377 385 352 TO DEVD + leupeptine 293 247 284 292 Example III Study of the action of protease inhibitors on ervthrocvtes, under storage conditions transfusion centers. The blood was collected at the Regional Center for Blood Transfusion in Lille, under the usual conditions, in "collection medium" at pH 5.6. He was leukoreduced and depleted. The erythrocytes are transferred to a "storage medium" at neutral pH, with

a final hematocrit of 30%.

  Erythrocytes are normally stored at 4 C.

  For the present experiment, an aging has been carried out

  accelerated by incubating them at 37 C for 3 and 4 days.

  At the same time, aliquots are incubated in the presence of

  inhibitors at 3 mM concentration.

  After 3 and 4 days, the externalization of the phosphatidylserine is measured, as in Example I. Table VI shows the inhibition of the externalization of the phosphatidylserine under the action

inhibitors.

Table VI

  Percentage inhibition of phosphatidylserine outsourcing Inhibitor: 3 day 4 day incubation incubation

DEVD 58 42

YVAD 72 65

  Leupeptin 62 52 DEVD + Leupeptin 68 64

Claims (9)

  1. Composition for delaying senescence, self-destruction and hemolysis of erythrocytes, characterized in that it comprises inhibitors   specific to the active site of cysteine proteases.   2. Composition according to claim 1, characterized in that the inhibitors have a structural analogy with the peptide motif   specifically recognized by said cysteine proteases.   3. Composition according to claim 1 or 2, characterized in that the inhibitors have a structural analogy with a tetrapeptide motif   specifically recognized by caspases.   4. Composition according to claim 1 or 2, characterized in that the inhibitors have a structural analogy with a tripeptide motif   specifically recognized by calpains.   5. Composition according to claim 1 or 2, characterized in that it comprises a mixture of inhibitors analogous to the tetrapeptide motif according to claim 2 and inhibitors analogous to the motif   Tripeptide according to claim 3.   6. Composition according to any one of   claims 1, 2, 3 and 5, characterized in that the   inhibitors are chosen from Asp-Glu- tetrapeptides   Val-Asp, Tyr-Val-Ala-Asp, or the tripeptide Z-Val-Ala-Asp, or derivatives thereof modified upstream or downstream of the aforementioned specific sequence, and are used alone or in mixture.   7. Composition according to any one of   claims 1, 2, 4 and 5, characterized in that the   inhibitors are chosen from leupeptin or its derivatives and the molecule E-64 or its derivatives, used alone or mixed.   8. Method for delaying senescence, self-destruction and hemolysis of erythrocytes, characterized in that it comprises the addition of   Composition according to any one of Claims 1 to 7 in the medium for preserving erythocytes at a final concentration of between 0.1 and 100 mM.   9. Use of the composition according to any one of claims 1 to 7 for the preparation of   medicines intended for the treatment of pathologies linked to early or aberrant manifestations of senescence or self-destruction and hemolysis of erythrocytes.