WO1996030026A1

WO1996030026A1 - Pharmaceutical compositions for preventing and treating malaria, based on a member of the chondroitin-glycosaminoglycan family

Info

Publication number: WO1996030026A1
Application number: PCT/FR1996/000458
Authority: WO
Inventors: Jürg Gysin; Bruno Pouvelle; Artur Scherf
Original assignee: Institut Pasteur
Priority date: 1995-03-27
Filing date: 1996-03-27
Publication date: 1996-10-03
Also published as: FR2732219A1; FR2732219B1; AU5337596A

Abstract

A pharmaceutical composition for treating or preventing malaria-related complications, characterised in that it contains a physiologically active agent consisting of chondroitin-4-sulphate.

Description

PHARMACEUTICAL COMPOSITIONS FOR THE PREVENTION AND TREATMENT OF MALARIA. BASED ON A MEMBER OF THE CHONDROIN-GLYCOSAMINOGLYCAN FAMILY

The present invention relates to malaria, in particular means for the therapy or prevention of malaria, and more particularly when the etiological agent responsible for the disease is Plasmodium falciparum. The sequestration of erythrocytes infected with Plasmodium falciparum in the microvascular network of the various organs plays an essential role in the pathogenicity of severe Plasmodium falciparum infections. (1, 2, 3). A histopathological study has indeed revealed that sequestrations of Plasmodium falciparum were sometimes observed in the cerebral micro-vascularization in Saimiri sciureus. one of the primates used frequently as animal models for the study of the evolution in man of malaria, more particularly of the forms of this disease induced by parasites of the Plasmodium falciparum type.

It has in fact been noted that neurological complications with fatal outcome can occur in the primate Saimiri sciureus ariotype 14-7, parasitized by different isolates of P.falciparum and that they can occur at different times in the parasite evolution, without apparent correlation with the duration or degree of parasitaemia, which is comparable to observations made in humans, especially when the latter is suffering from malaria or subject to pernicious attacks.

The histopathological studies carried out in the Saimiri having succumbed to a pernicious access have shown a great similarity with those which have been described in humans. Our results show that the vascular obstructions at the level of the microvessels of the brain are discontinuous and that their topography is very variable in this host, which is similar to the observations more recently made in humans who have died of malaria and those made in the model. experimental cerebral malaria Macaca mulatta / P.coatnevi. At the Saimiri as in humans, the vascular obstructions are often more important and sometimes almost exclusively localized at the level of the cerebellum, causing significant circulatory discomfort of the blood flow, which can alone explain the comatose state and the death of the patient or the 'animal.

The reactivity of various cell types with erythrocytes infected with Plasmodium falciparum. and the capacity of a certain number of monoclonal antibodies described in the literature (2.1 1) to interfere with this reactivity, is at the origin of the hypothesis already formulated that this process would be mediated, on the one hand , by specific adhesion ligands present on the surface of infected erytrocytes and, on the other hand, by receptors which are expressed on the surface of endothelial cells (2, 4, 5, 6, 7).

The invention is based on the identification of a new adhesion receptor which is expressed on the surface of endothelial cells originating from micro-vessels of Saimiri brains and constituted in particular by chondroitin-4 sulfate. In the course of the experiments which led to the present invention, two monoclonal antibodies directed against this new receptor were developed. They have been shown to be able to effectively block the adhesion of erythrocytes infected by several strains of the parasite to endothelial cells, whether these are of Saimiri brain origin or human pulmonary. The ability of these monoclonal antibodies to attach to endothelial cells has also been specifically inhibited by chondrotin-4-sulfate (CSA). Similarly, the pretreatment of endothelial cells with chondroitinase ABC and AC abolishes the capacity of infected erythrocytes to attach to these endothelial cells, hence the conclusion reached by the inventors, namely that an endothelial receptor for the adhesion of erythrocytes infected with Plasmodium falciparum. contained a member of the chondroitin-glycosaminoglycans family, more specifically chondroitin-4-sulfate (CSA)

In fact, the inventors have shown that intramuscular and oral administration of CSA to the Saimiri monkey completely inhibits the cytoadherence of erythrocytes infected with Plasmodium falciparum to endothelial cells of the brain. The object of the invention is therefore to provide pharmaceutical compositions capable of inhibiting the adhesion of erythrocytes infected by malarial parasites, more particularly of the Plasmodium falciparum type to endothelial cells. Such a composition is characterized in that it contains a physiologically active agent which competes with a receptor carried by the endothelial cells of the host infectable by P. falciparum and containing a member of the family of chondroitin-glycosaminoglycans, in particular of chondroitin-4-sulfate, this receptor allowing the adhesion of erythrocytes, when these are infected with Plasmodium falciparum. to these endothelial cells. According to another aspect of the invention, the pharmaceutical composition is characterized in that it contains a physiologically active agent which comprises a structure based on chondroitin-4-sulfate (CSA).

It is understood that the expression "chondroitin-4 sulfate", as used in the present text, relates both to chondroitin-4 sulfuric acid in its acid forms and to its therapeutically acceptable salts, in particular alkaline salts or alkaline earth, of this acid form.

Although the presence of CSA is preferred, the inventors have established that the inhibition of the adhesion of infected erythrocytes to the endothelial receptor is undoubtedly a function of the charge of the inhibitor molecule. Therefore, other molecules functionally analogous to CSA having a charge distribution similar to CSA can also be used. It goes without saying that the free reactive groups, in particular free hydroxyl carboxyl carried by the saccharide units contained in the chondroitin group of these chondroitin-4 sulfates can be substituted by particular chemical groups, since these substitution groups do not alter the ability of the substituted chondroitin-4 sulfates to compete with the relevant endothelial cell receptor. Preferably, the active agent of the composition according to the invention is an oligomer containing the structure

the number of monomer units contained in this oligomer being sufficient to prevent the attachment of P. falciparum to the cell receptor. According to another particular embodiment of the invention, the composition contains at least 90% of CSA of the chondroitin sulfates present, or even in a practically pure state. But this is not an essential condition. It is indeed also possible to have recourse, for the above-mentioned therapeutic applications, to mixtures, for example natural, of chondroitin sulphates, since at least part of these chondroitin sulphates consist of chondroitin-4 sulphate. Mention will be made, by way of examples, of the composition based on sulfuric acid chondroitin (sodium salt) sold under the brand STRUCTUM (Dictionary VTDAL 1993, page 1333, Editions du VIDAL) and used to date in adjunct treatment arthritis pain The invention also relates to the compositions described above for the treatment or prevention of malaria, more particularly complications of malaria involving the sequestration of malaria parasites, in particular Plasmodium falciparum. even more targeted for the treatment or prevention of pernicious accidents caused by Plasmodium falciparum. and involving the adhesion of erythrocytes infected by this parasite to endothelial cells. We know, in fact, that this type of accident results, among other things, from phenomena that are similar to those characteristic of thromboses and that, more particularly, at the level of cerebral or pulmonary micro-vascularization. This same type of accident is likely to be observed in other locations, for example, in the cardiac micro-vascularization It is naturally the clinician's responsibility to adapt the effective doses of the active ingredient, in particular chondroitin-4-sulfate, to suit the patient, which must be administered to him either for preventive or curative purposes. By way of illustration only, it will be mentioned that appropriate ranges of active doses calculated on the basis of chondroitin-4-sulfate, which can be administered in particular parenterally, are from 1 to 100 mg / kg of body, in particular from 5 to 10 mg / kg intramuscularly for curative purposes, these doses may be 5 to 10 times lower when administered for preventive purposes. In the case of oral administration, the appropriate doses are of the order of 5 times higher than the corresponding doses proposed above for parenteral administration.

Other characteristics of the invention will also appear on the occasion of the description of the biological tests which follow.

In order to study the effect of the parameters involved in the sequestration phenomenon of Plasmodium falciparum in the microvascular network of the brain of the monkey Saimiri sciureus kariotype 14-7, two clones of monoclonal antibodies interfering with the capacity of infected erythrocytes were produced. by P.falciparum to adhere to five clones of Sothiri brain microvessel endothelial cells (CECS), using the techniques described in the prior art (12) (13). The capacity of these monoclonal antibodies, designated 1 G1 1/1 C7 and 4B2 / 1B4, which have been shown to react specifically with the surface of CECS, has been abolished when brought into contact with endothelial cells, presence of chondroitin-4-sulfate. These antibodies have the capacity to inhibit the adhesion of parasitic strains of Plasmodium falciparum. Palo-AJto (FUP) l and IPL / BRE 1 to the clone of CECS Se 1707 (93 to 97% inhibition) and, admittedly in a more variable manner, (4 to 77%) with other isolates of the parasite.

The evaluation of the adhesion of the monoclonal antibodies (ac.m) 1G1 1 / 1C7 (lgGl) and 4B2 / 1B4 (IgM) to the CECS was carried out by incubating the confluent CECS on slides (12 dots IFA slides) during 1 hour with 1-10 μg ml of each of the monoclonal antibodies. Although inhibition of the adhesion of infected erythrocytes in the presence of one of the two monoclonal antibodies was very effective, the dissociation of infected erythrocytes attached to CECS was, in general, only partial (<20%), when they were used at doses of 10 μg of monoclonal antibodies (ac.m.) / ml.

The responsiveness of ac.m. IG1 1 / 1C7 and 4B2 / 1B4 with the CECS was determined by the "FACScan" device sold under the brand BECKMAN. The relative fluorescence intensities of each of the clones incubated with the 2 mAbs. have been measured. The immunostaining of CECS by the 2 ac.m., revealed by transmission electron microscopy with colloidal gold, made it possible to highlight the presence of their antigens on the surface of endothelial cell membranes, among micro- villi and pseudopodia. When immunostaining is performed on CECS with cytoadherent infected erythrocytes, gold particles are observed exclusively at the points of contact between the membranes of endothelial cells and the protuberances (in English "knobs") carried by the erythrocytes.

In order to analyze the nature of the new adhesion receptor, five CECS clones were treated with trypsin, but this treatment did not abolish their recognition by 1G1 1 / 1C7 and 4B2 / 1B4 to CECS. However, the treatment of these clones with chondroitinase ABC (lU / ml for 60 n) or chondroitinase AC, but not with chondroitinase B, abolished the adhesion of erythrocytes infected with Palo Alto (FUP) l and IPL BRE1 almost completely (86-99%). This result shows that a member of the chondroitin-glycosaminoglycan family, in this case chondroitin-4 sulfate (CSA), is involved in the adhesion of infected erythrocytes to endothelial cells. It corresponds to the new adhesion receptor of infected erythrocytes. The coincubation of 5 × 10 ⁶ infected erythrocytes / ml with 1 mg / ml of CSA (and not with other chondroitin sulfates: CSB or CSC) on confluent CECS resulted in approximately 50% inhibition of adhesion of erythrocytes infected either with Palo -Alto (FUP) l either by IPL BRE1, but did not significantly alter the adhesion of erythrocytes infected with the clone TM 284/62 (Table 1). Similar results have been obtained with isolates derived from patients with severe malaria. Two of the six isolates showed pronounced cytoadherence dependent on the CSA, so that the presence of the molecule adhering to the CSA on the surface of infected erythrocytes. The presence of CSA on the CECS was confirmed by analysis in the "FACScan" apparatus using the anti-chondroitin-4-sulfate mouse monoclonal antibody (MAB 2030, marketed by Chemicon International Inc, Temecula, CA 92590) and an anti-chondroitin-rabbit sulfate polyclonal serum (AB 1918, marketed by Chemicon International Inc, Temecula, CA 92590).

In order to determine that the CSA is the target molecule of the monoclonal antibodies 1G11 / 1C7 and 4B2 / 1B4, the inventors carried out inhibition studies. By incubating 10 μg of 1G1 1 / 1C7 or 4B2 / 1B4 with an excess of 2 mg / ml of CSA, the adhesion of these antibodies to Se 1707 (a clone of endothelial cells of the microvascular network of Saimiri brain which did not express none of the parasite's known adhesion receptors) was significantly decreased. The question then arose as to whether these results with CECS could be extended to human endothelial cells. To this end, primo explant endothelial cells were obtained from human lungs (CEPH). These cells reacted with mAbs. IGl 1 / 1C7 and 4B2 / 1B4 and with anti-chondroitin sulfate antibodies (AB 1918). Finally, it was possible to similarly inhibit the adhesion of erythrocytes infected with Plasmodium falciparum (Palo Alto / FUP 1, IPL / BRE1 strains) to these cells by the CSA, and by treatment with the drug to base of chondroitin-4-sulfate known under the brand STRUCTUM (approximately 80% inhibition in the presence for 1 mg / ml of the drug STRUCTUM). Inoculation of monkeys with 50 mg of CSA resulted in bioavailability of CSA, for at least 8 hours, allowing inhibition (> 92%) of the adhesion of infected erythrocytes to CECS (Tables 2 and 3)

The invention thus makes it possible to obtain particularly active drugs for preventing or treating the neurological complications of a P. falciparum infection. In particular, it is intended for the prevention or treatment of pathological biochemical phenomena which seem to intervene in cascade and probably following vascular obstructions due to sequestrated parasitized red blood cells. The invention also relates to drug combinations or combinations involving the CSA on the one hand, and an antimalarial drug, such as quinine or drugs known under the brands (by way of example) "CHLOROQUINE", "MEFLOQUINE", "AMODIAQUINE", etc. In the case of combinations, it will immediately appear to those skilled in the art, that the two constituents can be coupled together, for example via one of the reactive functions (hydroxyl or carboxyl groups) carried by the saccharide groups included in chondroitin-4 sulfate and amino functions carried by antiparasitic drugs, at least when they are behave. It will be appreciated that under these conditions, the part of the antiparasitic drug will contribute to killing the parasites released into the blood circulation, in particular during the intervention of the chondroitin-4 sulphate part in the competition between the infected erythrocytes and the endothelial cells. . The invention is in no way limited to the use of compositions based on chondroitin-4 sulfate, for in vivo treatments. It can also be used outside the human body, in particular for an extra¬ bodily purification of blood, to rid it of parasitized erythrocytes. The invention therefore also relates to a method consisting in bringing the blood or a blood flow capable of containing parasitized erythrocytes into contact with chondroitin-4 sulfate, the latter preferably being fixed on a solid support, such as a resin suitable, for example, of a type sold under the brand SEPHADEX. This fixing can be carried out in any case per se as, for example, via covalent bonds between the resin and chondroitin-4 sulfate, it being understood of course that this bond must be such that it does not interfere with the ability of chondroitin-4 sulfate groups to fix erythrocytes parasitized by P.falciparum or similar parasites Table 1

CSA inhibition of the adhesion of P. falciparum-infected erythrocytes (IRBC) to Saimiri brain endothelial cells (SBEC) and human lung endothelial cells (HLEC) pretreated or not with an ABC chondroitinase

Inhibition of IRBC adhesion to SBEC and HLEC, when they are co-incubated with CSA (1 μg / ml) or after pre-treatment of SBEC with chondroitinase ABC (lU / l for 60 minutes). The results are expressed as% inhibition (triplicate tests). The adhesion of IRBC e the absence of CSA or of pretreatment with chrondroitinase is expressed in IRBC fixed mm ² on confluent SBECs. Table 2

Inhibition of the cytoadherence of erythrocytes parasitized by P. falciparum (IRBC), by the drug sold under the brand "STRUCTUM"

IRBC / mm ² - Inhibition by "Structum" in% compared to controls

Palo-Alto (FUP) l

Sc3A4 5581 92 + 2

SclD 3771 78 + 6

Sc93006 / C2 3510 77 + 7

Table 3

Inhibition of the cytoadherence of erythrocytes parasitized by P. falciparum (IRBC), by the bioavailable CSA in sera of Saimiri monkeys

IRBC / mm ² % Inhibition by CSA Sc34A Sc93006 / C2 Se 34A Sc93006 / C2

TO 4861 + 925 2671 + 423

TO + 30 '373 + 256 121 + 54 92 95

TO + lh 342 + 235 129 + 8 93 95

TO + 2h 317 + 208 94 + 27 93 96

TO + 4h 291 + 31 48 + 26 94 98

TO + 8h 267 + 144 40 ± 10 95 99

REFERENCES

1. Aikawa, M. (1988). Cerebral malaria. Am. J. Trop. Med. Hyg. 39, 3.

2. Howard, R.J. and Gilladoga, A D. (1989) Moleeular studies related to the pathogenesis of cerebral malaria. Blood. 74, 2603.

3. Sein, K.K., Brown, A.E., Maeno, Y., Smith, CD. Corcoran, K.D., Hansukjariya, P., Webster, H.K. and Aikawa, M. (1993b). Sequestration patttern of parasitized erythrocytes in cerebrum, midbrain, and cerebellum of Plasmodium coatneyi -mfected rhesus monkeys (Macaca mulatta). Am. J. Trop. Med. Hyg. 49, 513.

4. Sherwood, J.A., Roberts, D.D., Spitalnik, S.L., Marsh, K., Harvey, E.B., Miller, L.H. and Howard, R.J. (1989) Studies of the receptors on melanoma cells for Plasmodium falciparum infected erythrocytes. Am. J. Trop. Med. Hyg. 40, 1 19.

5. Ockenhouse, CF. and Chulay, J.D; 1988 Plasmodium falciparum sequestration. OKM5 antigen (CD36) mediates cytoadherence of parasitized erythrocytes to a myelomonocytes cell line. J. Infect. Say. 157.554-

6. Ockenhouse, CF., Tegoshi, T. Meano, Y., Benjamin, C, Ho, M, Aikawa, M and Lobb, RR (1992) Human vascular endothelial cell adhesion receptors for Plasmodium falapamm-infecied erythrocytes: roles of ELAM -1 and VCAM- 1. J. Exp. Med. 176, 1183.

7. Bamwell, J., Ockenhouse, C. and Knowles, D., (1985) Monoclonal antibody OKM5 inhibits the in vitro binding of Plasmodium falciparum-infected erythrocytes to monocytes, endothelial cells and C32 melanoma cells. J. Immunol. 135, 3494.

8. Berendt, A.R., Simmons, D.L., Tansey, J., Newbold, CI. and Marsh, K. (1989) Intercellular adhesion molecule- 1 is an endothelial cell adhesion receptor for Plasmodium falciparum. Nature. 341, 57.

9. Gareth, D., Turner, H., Morrison, H., Jones, M., Davis, TME, Looareesuwan. S., Buley, ID, Gatter, KC, Newbold, CL, Pukritayakamee, S., Nagachinta, B., White, NJ and Berendt, AR (1994). Year immunohistochemical study of the pathology of fatal malaria. Evidence for widespread endothelial activation and a potential role for intercellular adhesion molecule- 1 in cerebral sequestration. Am. J. Pathol 145, 1057

10. Gysin, J., Aikawa, M., Tourneur, N. and Tegoshi, T. (1992) Experimental Plasmodium falcipiarum cerebral malaria in the squirrel onkey Saimiri sciureus. Exp. Parasitol. 75, 390.

11. Gay, F., Robert, C. Pouvelle, B., Peyrol, S., Scherf, A. and Gysin, J. (1995). Isolation and characterization of brain vasculature endothelial cells from Saimiri monkeys: an in vitro model for Plasmodium falciparum-infected erythrocytes cytoadherence. J Imm. Meth (in press)

12. Gysin, J, Hommel, M and Pereira da Silva, L (1980) Experimental infection of the squirrel monkey Saimiri sciureus with Plasmodium falciparum J Parasitol. 66, 1003

13. Gysin, J. and Fandeur, T. (1983) Saimiri sciureus (Karyotype 14-7) an alternative experimental model of Plasmodium falciparum

14. Gysin, J. (1991) Relevance of the squirrel monkey as a model for experimental hu an malaria. Res. Immunol. Inst. Pastor. 142, 649.

15. Muanza, K., Gay, F. Behr, C. and Scherf. A .. Primary culture of human lung endothelial cells as an in vitro model for Plasmodium falciparum-m ^' fectQd erythrocytes cytoadherence. Infec. Immun.

(submitted)

Claims

1. Pharmaceutical composition containing at least one pharmaceutical vehicle, characterized in that it contains a physiologically active agent which competes with a receptor carried by endothelial cells and containing a member of the family of chondroitin-glycosaminoglycans, in particular chondroitin- 4-sulfate, this receptor authorizing adhesion to these endothelial cells of erythrocytes when they are infected with Plasmodium falciparum.

2. Pharmaceutical composition containing at least one pharmaceutical vehicle, characterized in that it contains a physiologically active agent which comprises a structure based on chondroitin-4-sulfate.

3. Composition according to claim 2, characterized in that the active agent contains an oligomer of the structure:

The number of monomer units contained in this oligomer being sufficient to allow attachment to the above-mentioned receptor.

4. Composition according to claim 2 or 3, characterized in that it contains at least 90% of chondroitin-4-sulfate relative to the chondroitin sulfates present

5. Composition according to any one of claims 2 to 4, characterized in that it contains chondroitin-4-sulfate in the practically pure state.

6. Composition according to any one of claims 1 to 3, characterized in that the physiologically active agent consists of a mixture of natural chondroitin-sulfates containing chondroitin -4 sulfate.

7. Composition according to any one of claims 1 to 6, characterized in that it also contains a separate antiparasitic drug, in association or in combination with the constituents chondroitin-4 sulfate.

8. Composition according to Claims 1 to 7 for the treatment or prevention of malaria, in particular of the complications of this disease which arise from malaria.

9. Composition according to claim 8, to prevent the sequestration of malaria parasites, in particular Plasmodium falciparum.

10. Composition according to claims 1 to 9, for the treatment or prevention of pernicious accidents induced by malaria parasites, in particular by P.falciparum. and involving the adhesion of erythrocytes infected by this parasite to endothelial cells.

11. Use of a physiologically active agent as defined in claims 1 to 9, for obtaining a medicament intended for a therapeutic or preventive use of malaria.

12. Use according to claim 1 1, characterized in that the medicament is intended to prevent the sequestration of malaria parasites, in particular of P.falciparum.

13. Use according to claim 11 or claim 12, characterized in that the medicament is intended to treat or prevent pernicious accidents or various complications induced by malaria parasites, in particular by P.falciparum. and involving the adhesion of erythrocytes infected by this parasite to endothelial cells