FR2722991A1 - USE OF BETA2-GLYCOPROTEIN I IN AT LEAST ONE OF ITS FORMS AS ANTI-INFECTIVE AGENT AND CORRESPONDING PHARMACEUTICAL COMPOSITION - Google Patents

Abstract

Use of the beta 2-glycoprotein I, in at least one of its forms, for producing an anti-infectious agent for treating infectious diseases and capable of in vitro and in vivo activity. Used in vivo, it provides an anti-infectious drug for the medical treatment of animals or humans.

Description

USE OF ss2-GLYCOPROTEIN I UNDER AT LEAST
ONE OF ITS FORMS AS ANTI-INFECTIOUS AGENT AND
CORRESPONDING PHARMACEUTICAL COMPOSITION.

 The invention relates to the use of a plasma protein as an anti-infective agent and an injectable pharmaceutical composition containing said protein.

 Ss2-glycoprotein I is a plasma glycoprotein, which has notably been sequenced in the articles of J. LOZIER et al.

Proc. Natl. Acad. Sci. ISA, Vol. 81, pp. 3640-3644, July 1984 and T. KRISTENSEN et al., FEBS Letters, Vol. 289, 1991, pages 183-186. B2-glycoprotein I is also called Apolipoprotein
H (abbreviated ApoH). The first 20 amino acids of this glycoprotein from the N-terminus are the following: Gly-Arg-Thr-Cys-Pro-Lys-Pro-Asp-Asp-Leu-Pro-Phe-Ser-Thre-Val-
Val-Pro-Leu-Lys-Thre.

 One form of ss-2 glycoprotein I, namely ss-2 'glycoprotein I, is described in the French patent application No. 93 01 399 filed by the applicants on February 9, 1993 under the title "Process for obtaining an aqueous protein composition, corresponding composition, contained glycoprotein and its use ".

This ss2'-glycoprotein I is characterized by its isolation from the residue fixed on a column of affinity chromatography on a gel carrying sulphate groups, used in a process for purifying the albumin of the blood plasma described in FR-A -2,690,444. B2'-glycoprotein I differs from B2-glycoprotein I in that, in the region between amino acids 315 to 321, which has been described by J. LOZIER et al (see reference below). above), a difference of an amino acid appears: it is the presence of a serine instead of a threonine. According to this document, the albumin is separated from the other proteins by a liquid chromatographic method, in which the aqueous solution containing the albumin is passed into at least one so-called "hydrophobic" chromatography column filled with a suitable particulate material. to retain some of the proteins other than albumin; to complete the separation, it is also proposed to pass the aqueous albumin solution in at least one affinity chromatography column containing a particulate carrier neutral or close to neutrality charged with a polysulfated compound. The effluent obtained by this process consists of a purified albumin solution, the majority of proteins other than albumin being fixed, either on the hydrophobic chromatography column (s) or on the column (s) of affinity chromatography. By subjecting the support of the aforementioned affinity chromatography column (s) to elution, preferably elution by increasing the ionic strength, a protein composition is obtained, the protein content of which comprises at least one less 5% by weight of ss2'-glycoprotein I and from which the B2'-glycoprotein I can be purified.

 The French patent application 93 01 400 filed on February 9, 1994 entitled "Method for detecting and / or assaying viral compounds and supporting a carrier glycoprotein" shows that certain viral compounds bind to B2'-glycoprotein I and described a method for detecting and assaying viral compounds with B2'-glycoprotein I.

 According to the present invention, it has now been found that, unexpectedly, B2-glycoprotein I has an anti-infective effect. An object of the invention is therefore the use of B2-glycoprotein I for the preparation of medicaments for combating infectious conditions.

The infectious disease can be caused in particular by a microbe such as a bacterium, a virus, a parasite or a fungus.

 In particular, it has been found that B2 glycoprotein I can be used for the treatment of an infectious disease included in the group formed by hepatitis B, herpes simplex, the immune deficiency generated by HIV1, HIV2 or SIV, leishmaniasis, malaria, toxoplasmosis, amoebiasis, candidiasis, aspergillosis, borellioses.

B2-glycoprotein I can be used in the form of
B2'-glycoprotein I described above. It can be implemented in the form of a protein composition, preferably the aqueous protein composition obtained by elution of an affinity chromatography column in the process for purifying human albumin described in FR-A-2,690. 444.

 The present invention also relates to a pharmaceutical composition containing, in a pharmaceutically acceptable carrier, at least one anti-infectious agent containing an active amount of B2 'glycoprotein I in at least one of its forms.

 In the case of treatment of mammals, particularly humans, a total dose of 8 to 80 mg of active protein per kg of treated subject is suitable in an injection treatment. However, higher doses can be used as long as no undesirable effects are observed.

The examples given below show the antiinfective effect of ss2'-glycoprotein I. In the tests carried out, the infection of BALB / C mice by Leishmania Mexicana was followed in vivo.
Amazonensis, hereinafter referred to as LMA, human parasite infecting macrophages (Mc ELRATH MJ, KAPLAN G., MUSVAT A. and
COHN, ZA Cutaneous Leishmaniasis. The defect in T. cell influx in
BALB / c mice. J. Exp. Med (1987) 165 p. 546).

Preparation of B2'-glycoprotein I
The starting material used is a fractionated human plasma according to the method described by KISTLER AND
NITSCHMAN (Vox Blood 7, 414-424 (1962)), which is a method derived from that of COHN.

Supernatant IV, which comprises 40% (by volume) of ethanol and which has a pH of 5.85 ± 0.05, is used, the supernatant is half-diluted with a solution of NaCl at 7 g / l. The pH is adjusted to 7.45
+ 0.05 with a 1N sodium hydroxide solution. The albumen is preconcentrated up to 90 g / l in an "Omega" type ultrafiltration cassette (Filtron) used in an ultrafiltration system "Minisette SS
Cell N7r Cell "(Filtron Techn Corp.) This cassette has a filtering surface of 0.07 m2 and a retention threshold of 30 KDa.Circulation is provided by a peristaltic pump at a pressure of 2 x 105 Pa at the beginning of the operation at 5 x 105 Pa towards the end When the concentration of 90 g / l of albumin has been reached, a solution of NaCl at 9 g / l is added to the albumin solution and continued to dialyze at constant volume until an ethanol level of less than 0.1% by volume is obtained.When the ethanol has thus been removed, the dialysis is continued by concentrating the solution to 200 g of albumin per ml. liter.

The pH is adjusted to 7.10 ± 0.05 with a 1N hydrochloric acid solution.

 The albumin solution thus prepared from the supernatant IV is then sterile filtered ("Millex-GS" 0.2 micron filter (Millipore)).

 The crude aqueous albumin solution obtained first undergoes affinity chromatography. This chromatography is carried out in a 50 ml column (2.5 cm × 10 cm) (Biorad), loaded with a particulate material sold by the company "SIGMA" under the trade name "DEXTRAN BEADS SULFATED". The column is previously equilibrated by passing in the bed 3 column volumes of physiological saline. The albumin solution is then sent on this column.

 The progress of the chromatography is monitored by measuring the optical density at 280 nm of the column output fractions. The flow rate of the column is 16 cm / hour; the chromatography is carried out between 20 and 25 C.

 After passage of the albumin solution, the affinity column is washed with a buffer consisting of mono- and disodium phosphates at 0.01 mol / l, and sodium chloride at 0.15 mol / l having a pH of 7.00 + 0.05 until the optical density of the effluent is less than 0.1. The proteins fixed on the affinity column are then eluted, by increasing the ionic strength, by passing a 2M solution of NaCl. The washing and the elution are carried out at room temperature, the washing and elution rates being 16 cm / h.

 SDS-Page denaturing electrophoresis was performed on the obtained solution. An aqueous composition was obtained containing 55% by weight of B2'-glycoprotein I relative to the total proteins.

The ss2'-glycoprotein I is then separated and purified as follows: the protein composition obtained by elution of the affinity chromatography column using a saline solution of
NaCl 2 mol / liter, is diluted 10 times in a buffer consisting of mono- and disodium phosphates, especially at 0.01 mol / liter, in proportions giving a pH of 6.8 + 0.05. It is then subjected to phosphate gel chromatography. This chromatography is carried out in a 50 ml column (2.5 cm × 10 cm) (Biorad), loaded with a particulate material sold by the company "BIGRAD" under the trade name "BIO-GEL HTP". The column is pre-equilibrated by passing through the bed 3 column volumes of buffer consisting of mono- and disodium phosphates, especially at 0.01 mol / liter, in proportions giving a pH of 6.8 + 0.05. The protein eluate from the previous chromatography, diluted, is then sent onto this column. The chromatography is carried out with a flow rate of about 15 cm / h at a temperature of 20 C. The effluent is removed and the chromatographic support is then subjected to a washing carried out using a phosphate buffer, identical to that which has served to balance the column. Washing is maintained as long as the optical density of the effluent is greater than a predetermined value, for example 0.1 ODU (optical density unit).

 The B2'-glycoprotein I which binds to the phosphate gel is then eluted by increasing the ionic strength, in particular by means of a KCl solution having a concentration close to 1M. This elution solution consists of mono- and disodium phosphates, especially at 0.01 mol / liter, in proportions giving a pH of 6.8 + 0.05 and KCl 1 mol / liter. The progress of the chromatography is monitored by measuring the optical density at 280 nm of the column output fractions. The balancing flow of the column, charging, washing and elution is 16 cm / h. Chromatography is performed at 200 C.

 The eluate is recovered and then dialysed, preferably in injectable water. The isolated B2'-glycoprotein I is then lyophilized and stored at -25 ° C. At the time of use, the desired dose is dissolved in physiological saline (0.15 M NaCl).

 Various explanations can be suggested on the origin of the unexpected anti-infective effect of ss2'-glycoprotein I, it being understood that these hypotheses can not, under any circumstances, be considered as likely to limit the invention.

We can, first of all, think of a recognition of the no-self. This could be confirmed by the link of the
B2'-glycoprotein I with viral compounds which is described in the French patent application 93/01 400. The direct link between
B2-glycoprotein I and HBV surface antigen was confirmed (HAIDER MEHDI, MJ KAPLAN, FY ANLAR, X. YANG, R.

BAYER, K. SUTHERLAND and ME PEEPLES, Hepatitis B Virus
Antigen Surface Binds to Apolipoprotein HJ of Virol (1994) 68, p.

2415-2424).

In addition, the fixation of the
B2'-glycoprotein I on a number of pathogens fixed on slide by cold acetone. In each case, the slide is washed with PBS buffer, after which the preparation is brought into contact with a
B2'-glycoprotein labeled with a fluorescent compound. This contact is maintained for 30 minutes at room temperature and is carried out with aqueous compositions respectively containing 2 μg / ml, 20 μg / ml and 100 μg / ml of protein. Two washes are then carried out for 5 min each with PBS buffer and the slides thus obtained are observed under an ultraviolet microscope. Positive results are obtained with the following infectious agents - 4 protozoa
Entamoeba histolytica, Toxoplasma gondii, Plasmodium falciparum, all three positive up to the concentration of 20 μg / ml;
Leishmania infantum positive up to the concentration of 2 yg / ml.

- 2 strains of fungi: Candida krusei, Aspergillus fumigatus positive up to the concentration of 100 llg / ml.

This experiment shows the link of B2 '-glycoprotein I with non-self structures other than viruses and allows to consider that the anti-infectious activity observed below with the
Leishmania extends to other pathogens such as those mentioned above.

 In the second place, it may be thought that B2'-glycoprotein I is involved in the defense of the body, its plasma concentration may be reduced during infection, especially during viral hepatitis (MOZER C., H. GEIGER , D. FEIST, Quantitative determination of single serum proteins during acute hepatitis in chlidhood, Eur J of Pediatria (1978), 128, pp. 123-128).

Moreover, it has a sequence homology with the "virokine", also called "secretory major protein" of the vaccinia virus (KOTWAL GJ MOSS B: Vaccinia virus encodes a secretory polypeptide structurally related to complement control proteins Nature (1988) 335, pp. 176-178). Several articles have shown that certain viral proteins are virulence factors because they have a blocking effect on non-specific defenses of the body. Thus, the Herpes Simplex Virus (HSV) C1 glycoprotein functions as a C3b receptor on virus-infected cells and inhibits complement lysis (Harris, SL, Franck
I., Yee A., Cohen GH, Eisenberg RJ and Friedman HM,
Glycoprotein C of Herpes Simplex Virus type 1 prevents complement mediated cell lysis and virus neutralization. J. Infect. Dis. (1990) 162, p. 331-337). Similarly, Epstein-Barr virus (EBV) functions as a third complement component receptor (Mold C.,
Bradt MB Nemerow GR, Cooper MR, Epstein-Barr virus regulates activation and processing of the third component of complement, J. Exp. Med. (1988) 168, p. 949-969). Herpes Virus
Saimiri codes for a protein called CCPH (Complement
Control Protein Homolog) which shows significant sequence homology with regulatory proteins known to interact with C3b and C4b; this virus also codes for another protein that shows 48% homology with a protein regulating the action of the
C9 (Hu CD 59) (RP Rother, Rollins SA, Fodor WL, Albrecht
JC, Setter E., Fleckenstrein B. and Squinto SP, Inhibition of
Complement Mediated Cytolysis by the Terminal Complement
Inhibitor of Saimiri Herpes Virus, J. of Virol. (1994), 68, p. 730-737).

Virokine is known to be an important component for virulence in vivo (Chang P., Lai AC, Pogo BG, Attenuated deletion mutant of vaccinia virus IHD.W recovered virulence by reinsertion of a terminal restriction fragment, Microbiol Pathogenesis (1992) , 13, p.

49-59). Its action is, at least partially, explained by the homologies of sequences (38%) with the first half of the gene of the
C4BP and by its binding with C3b and C4b which causes, in vitro, the inhibition of the complement action (Mc Kenzie R., Kotwal GP,
Moss B., Hammer CH, Frank MM, Control of complementary activity by vaccinia virus complement control protein, J. of Infect. Dis.

(1992), 166, p. 1245-1250). Sequence homology between virokine and B2-glycoprotein I could also explain this virulence if it has a role in the defense of the body.

Thirdly, the anti-infective effect could be due to its affinity for phosphorylated lipids. Indeed, in Malaria, the phospholipids released by Plasmodium have a toxic hypoglycemic action and are capable of inducing the release by macrophages of "tumor necrosis factor" (TNF). Ss2'-glycoprotein I is able to bind these phospholipids, inhibiting their harmfulness (K. TAYLOR, C. BATE C. AW, CAN RE, BUTCHER GA,
TAVERNE J., PLAYSAIR JHL Phospholipid containing toxic malaria antigens induce hypoglycemia, Clin. Exp. Immunol., (1992) 9Q, p. 1-5).

 Fourth, the anti-infective effect could be due to the interaction of B2'-glycoprotein I on Heparan sulfate. Indeed, the increased production of Heparan sulfate in tissues in a state of chronic inflammation can cause the production by macrophages of prostaglandin E2 (PG E2) or other substances likely to slow down the immune reaction (NS IHRCKE, THE

WRENSHALL, BJ LINDMAN and JL PLATT, Role of Heparan sulfate in immune system blood vessel interactions, Immunol. Today (1993) 14, P. 500-505); however, it has been reported that B2-glycoprotein I is capable of binding to Heparan sulfate (E. PLZE, WURM H.,
KOSTNER GM Investigations on B2-glycoprotein I in rat isolation from serum and demonstration in lipoprotein density fraction.

Int. J. of Biochem (1979) 11, p. 265-270). An inhibition of the action of Heparan sulfate by ss2'-glycoprotein I is therefore possible and could account for the anti-infectious action of this protein.

EXAMPLE I
Experimental protocol
A dose of LMA amastigotes obtained by in vitro culture is injected subcutaneously into the pads of BALB / C mice according to the article by Mc ELRATH et al. Cited above.

The dose of amastigotes is mixed with 25 μl of RPMI 1640 culture medium. The diameter of the paws, which increases with the progression of the infection, is measured at different times, using a vernier caliper.

4 BALB / C female 5-week-old mice weighing between 10 and 25 g were observed - a control mouse (ST) was neither infected nor treated with ss2'-
glycoprotein I, - an uninfected control mouse (SD) receives in the hind paw
30 μl of a solution of 132'-glycoprotein I in the serum
physiological at 1 mg / ml (ie 30 μg), - a mouse (SL) receives in each of the two hind legs 500,000
amastigotes of LMA, - a mouse (SDL) receives simultaneously
- in the left hind paw (SDL / PG) 500 000 amastigotes of
LMA and 30 μl of physiological saline,
- in the right hind paw (SDL / PD) 500 000 amastigotes of
LMA and 30 μl of a solution of 132'-glycoprotein I at 1 mg / ml
(ie 30 Zg) -
Results
The diameters of the tabs, measured in mm, are given in Table I below. The appended FIG. 1 shows the evolution over time of the average diameter of the hind legs for each of the mice
ST, SD and SL. The evolution of the diameter of each hind leg of the SDL mouse is given for the right leg SDL / PD and for the left leg SDL / PG.

TABLE I

<tb> JPI <SEP> SDL / PD <SEP> SDL / PG <SEP> SL <SEP> SD <SEP> ST
<tb><SEP> 0 <SEP> 2.1 <SEP> 2,3 <SEP> 2,2 <SEP> 2,3 <SEP> 2,2
<tb> 17 <SEP> 2 <SEP> 2.5 <SEP> 2.4 <SEP> 2.3 <SEP> 2.1
<tb> 20 <SEP> 2,3 <SEP> 2,7 <SEP> 2,6 <SEP> 2,3 <SEP> 2,2
<tb> 23 <SEP> 2.6 <SEP> 2.4 <SEP> 2.8 <SEP> 2.4 <SEP> 2,3
<tb> 41 <SEP> 4.9 <SEP> 3.5 <SEP> 4.3 <SEP> 2.3 <SEP> 2.2
<tb> 54 <SEP> 4.4 <SEP> 4.1 <SEP> 5.4 <SEP> 2.3 <SEP> 2,3
<tb> 71 <SEP> 4.5 <SEP> o <SEP><SEP> 4 <SEP> 7.1 <SEP> 2.4 <SEP> 2,3
<tb> 86 <SEP> 2.8 <SEP> 5 <SEP> 8.9 <SEP> 2.4 <SEP> 2.4
<tb> JPI = days post-infection
The results show: 1) that there is no significant difference between the diameters of the
paws of ST and SD mice, which shows that B2'-glycoprotein I
alone does not cause measurable inflammation 2) an increasing increase in the diameter of the SL mouse paws
infected and untreated by 132'-glycoprotein I, which shows
infectivity of the LMA inoculum used 3) a small increase in the diameter of the right paw of the mouse
SDL (SDL / PD), especially after the 54th day; 4) an increase in the diameter of the left paw of the SDL mouse
(SDL / PG) which is significantly lower than in the case of the mouse
SL, which shows that the injected 132'-glycoprotein I only
in the right paw (SDL / PD) broadcast and acted remotely on the
Infectious focus of the left paw (SDL / PG).

To confirm the diffusion of ss2'-glycoprotein I, it was realized
an intraperitoneal injection of 500 μg of 132'-glycoprotein I in
the SDL mouse 71 days after infection. A phenomenon of necrosis
localized and well-defined right paw (SDL / PD) was observed
after 4 days, followed by retraction of the necrotic area after 6
days. By the 11th day, the underlying scar tissue was visible, the
diameter of the right paw being reduced (2.8 mm at the 86th day).

Peritoneal injection of B2'-glycoprotein I thus acted on
infection in the right paw.

EXAMPLE 2
Experimental protocol
It is the same as in Example 1 except that
a) the infection was carried out by injection of 10 million LMA promastigotes in each hind paw of 10 adult male BALB / C mice; 2 control mice were not infected.

 b) 132'-glycoprotein I was injected intraperitoneally in varying concentrations in a constant volume of 250 Ctl of saline.

1) 2 mice received 1 mg of B2'-glycoprotein I, each in two
intraperitoneal injections at 55 and 66 days after infection with
AML, 2) 1 mouse received 0.5 mg of 132'-glycoprotein I in one injection
intraperitoneal at 55 days, 3) 1 mouse received 0.1 mg of 132'-glycoprotein I in one injection
intraperitoneal at 55 days, 4) 1 mouse received 0.5 mg of 132'-glycoprotein I in 7 injections
daily intraperitoneal from 55 days after infection
by LMA, 5) 3 mice received 0.5 mg of 132'-glycoprotein I in 14 injections
daily intraperitoneal 35 yg starting 55 days after
infection, 6) for a positive control of the infection, 2 infected mice do not have
received B2'-glycoprotein I, 7) for a negative control, 2 uninfected mice received no
B2'-glycoprotein I.

 The increase in the average diameter of the legs from the day of the first injection of 132'-glycoprotein I, 56 days after infection with AML, as well as the standard deviations, are given in Table II below.

 The appended FIG. 2 shows the corresponding curves and the corresponding standard deviations.

 Since this test showed that identical results were obtained for 0.5 mg injections in small sequential doses for 7 or 14 days or as a single dose, the results were reported in the same column of Table II (D = 0). , 5 mg) below.

 In addition, no adverse effects were observed for the highest doses (D = 1 mg).

TABLE II

JPI <SEP> D = 1 <SEP> mg <SEP> D = 0.5 <SEP> mg <SEP> D = 0.1 <SEP> mg <SEP> D = O <SEP> Con
<tb><SEP> n = 2 <SEP> n = 5 <SEP> n = 1 <SEP> n = 2 <SEP> thrones <SEP>
<tb> 56 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> o <SEP><SEP> 0 <SEP> 0
<tb> 66 <SEP> 0.09 <SEP> + <SEP> 0.17 <SEP> 0.02 <SEP> + <SEP> 0.05 <SEP> 0.75 <SEP> + <SEP> 0.49 <SEP> 0.05 <SEP> + <SEP> 0.10 <SEP> 0
<tb> 78 <SEP> 0.08 <SEP> + <SEP> 0.26 <SEP> 0.04 <SEP> + <SEP> 0.09 <SEP> 1.10 <SEP> + <SEP> 0 , 28 <SEP> 0.053 <SEP> + <SEP> 0.19 <SEP> 0
<tb> 84 <SEP> 0.09 <SEP> + <SEP> 0.27 <SEP> 0.03 <SEP> + <SEP> 0.10 <SEP> 1.85 <SEP> + <SEP> 0 , 35 <SEP> 0.75 <SEP> + <SEP> 0.26 <SEP> 0
<Tb>

<tb><SEP> 91 <SEP> 0.13 <SEP> 0.32 <SEP> 0.01 <SEP> 0.013 <SEP> 2.15 <SEP> 0.07 <SEP> 0.93 <SEP> 0.05 <SEP> 0 <SEP>
<tb> JPI <SEP> = <SEP> Day <SEP> post <SEP> infection
<tb> D <SEP> = <SEP> dose <SEP> of <SEP>B2'-glycoprotein<SEP> I
<tb> n <SEP> = <SEP> number <SEP> of <SEP> mice
<Tb>
This table shows: 1) a markedly smaller increase in leg diameter at 84
and 91 days post-infection between mice treated with
1 mg and 0.5 mg and untreated or low-dose mice
(0,1 mg), 2) the existence, in this example, of a dose of maximum efficacy of
0.5 mg per mouse.

Claims (8)

 1 - Use of 132-glycoprotein I in at least one of its forms for obtaining a medicament for the treatment of infectious diseases.  2 - Use according to claim 1 for obtaining a medicament for the treatment of an infectious disease included in the group consisting of hepatitis B, herpes simplex, the immune deficiency generated by HIV1, HIV2 or SIV, leishmaniasis, malaria, toxoplasmosis, amoebiasis, candidiasis, aspergillosis, borellioses.  3 - Use according to claim 2 for obtaining a medicament for the treatment of leishmaniasis.  4 - Use according to one of claims 1 to 3, characterized in that it implements 132-glycoprotein I in its B2'-glycoprotein I form.  5 - Use according to one of claims 1 to 4, characterized in that 132-glycoprotein I is used in the form of an aqueous composition.  6-Use according to one of claims 1 to 5, characterized in that one uses a ss2-glycoprotein I extracted from human blood plasma.  7 - Pharmaceutical composition containing, in a pharmaceutically acceptable carrier, at least one antiinfective agent, characterized in that it contains, as an antiinfective agent, an active amount of 132-glycoprotein I in at least one of its forms.  8 - Composition according to claim 7, characterized in that it consists of an aqueous composition that can be administered by injection at a total dose of 8 to 80 mg of 132-glycoprotein I per kg of treated subject.