Chemistry: molecular biology and microbiology – Virus or bacteriophage – except for viral vector or... – Recovery or purification
Reexamination Certificate
1999-10-12
2004-10-19
Parkin, Jeffrey S. (Department: 1648)
Chemistry: molecular biology and microbiology
Virus or bacteriophage, except for viral vector or...
Recovery or purification
C435S235100, C435S068100, C435S005000, C530S350000
Reexamination Certificate
active
06806077
ABSTRACT:
The present invention relates to a method for screening substances capable of having a therapeutic action in the treatment of transmissible subacute spongiform encephalopathies (TSSEs) or so-called prion diseases, which comprises a step of isolating the PrPres from the spleen; the present invention also relates to methods for isolating the PrPres, which are particularly suited to the said screening method, and their applications in particular in the detection of PrPres.
Transmissible subacute spongiform encephalopathies are caused by nonconventional transmissible agents (NCTAs), also called prions, whose precise nature remains unknown to date. TSSEs comprise essentially Creutzfeldt-Jakob disease, in humans (CJD), scrapie, in sheep and goats, and bovine spongiform encephalopathy (BSE), in bovines; other encephalopathies have been demonstrated in mink or some wild animals such as red deer and elk.
The progression of these diseases is always fatal and there is currently no effective treatment.
In transmissible subacute spongiform encephalopathies, there is an accumulation of a host protein, PrP (or prion protein), in an abnormal form (PrPres), mainly in the central nervous system; PrPres copurifies with the infectivity and its accumulation precedes the appearance of histological lesions. In vitro, it is toxic for neuron cultures.
Two biochemical properties make it possible to distinguish PrPres from the normal PrP: PrPres is partially resistant to proteases and is insoluble in nonionic detergents such as Triton-X100.
The search for new molecules which may be effective in the treatment of these encephalopathies is hindered both by the absence of efficient in vitro models and the length of time for setting up in vivo experimental models, such as experimental scrapie in hamsters (80 to 365 days), or experimental scrapie in mice (180 to 550 days).
Consequently, the inventors set themselves the objective of providing an effective and reliable method of screening which does not exhibit the disadvantages of the experimental models currently used and which is more suitable for the requirements of practical use, in particular in that the evaluation of the action of the substances to be tested can be carried out in less than two months.
To do this, the inventors have found a reliable marker and have developed a reproducible protocol.
The subject of the present invention is a method for screening substances capable of having therapeutic action in the treatment of transmissible subacute spongiform encephalopathies (TSSEs or so-called prion diseases), characterized in that it comprises the following steps:
a) inoculation at time t
A
, into at least one laboratory animal such as a rodent, mouse or hamster (preferably several, divided into batches), by any appropriate route, of a nonconventional transmissible agent (NCTA) or prion;
b) administration to the said laboratory animal, by any appropriate route, of either a substance to be screened (test animal), or of a placebo (negative control animal), within a period between t
A
−15 days and t
C
, corresponding to the time when the PrPres level in the spleen of the said laboratory animal is at maximum or within a period between t
B
, corresponding to the time of the first detection of PrPres in the spleen of the said laboratory animal and t
C
; t
B
being between t
A
and t
A
+15 and t
C
being between t
A
+20 and t
A
+30, preferably between t
A
+25 and t
A
+30;
c) sacrificing of the animals within a time interval between t
B
and t
C
, preferably at t
C
, and collecting of the spleen;
d) isolation of the PrPres from the spleens collected, according to a suitable method of isolation comprising the homogenization of the spleen, followed by a specific extraction of the PrPres comprising a single separation step, from the homogenate obtained, and optionally the purification of the PrPres;
e) semiquantification of the PrPres obtained in step (d) by detection of the said PrPres by any appropriate method, producing a specific signal, followed by a comparison of the signal obtained with a calibration series of dilutions of a positive control consisting of a brain homogenate from an animal at the terminal stage of the disease; and
f) selection of the screened substances as a candidate for the treatment of transmissible subacute spongiform encephalopathies, if the PrPres, level obtained in the spleen of the test animal, in step e), is reduced by at least a factor of 2 compared with the level obtained under the same conditions with the negative control animal.
The times t
A
, t
B
and t
C
are expressed in days; t
A
=D
0
.
Indeed, the inventors have found, unexpectedly, that substances which increase the survival of infected animals, regardless of the mode of inoculation (peripheral or intracerebral), also result in a delay in the accumulation of PrPres in the spleen, detected under standardized conditions.
Standardized conditions are understood to mean, for the purposes of the present invention, conditions in which the following parameters are selected:
NCTA selected,
route of administration of NCTA,
method of isolation of the PrPres from the spleen.
For a strain selected from a given animal, at the terminal stage of the disease, the infectious titer is constant.
The detection of PrPres in the spleen makes it possible to observe much more rapidly the effects of the molecules to be tested, in particular the inhibition of the accumulation of PrPres, either within the hours following the inoculation (capture of the inoculum by the spleen and detection of a PrPres peak between t
A
and t
A
+1-2 days), or between 5 and 15 days after the infection; t
B
corresponds both to the detection of the peak at the time of capture of the inoculum and to the detection of the neosynthesized PrPres; this is the reason why t
B
is between t
A
and t
A
+15; these t
B
and t
C
values may vary within the ranges defined above, depending on the NCTA and the laboratory animal (mouse) selected; for example, when the UCTA corresponds to the murine strain C506M3 inoculated by the intraperitoneal route, in the C57BL/6 mouse, the neosynthesized PrPres may be detected in 100% of cases, from the 5th day post-infection (p.i.) (t
B
) and a plateau is observed from the 30th day p.i. (t
C
).
Such a method therefore makes it possible to select molecules capable of preventing the accumulation of PrPres; such molecules are considered to be capable of exhibiting therapeutic action in the treatment of TSSEs.
In accordance with the invention:
In step a):
the NCTA corresponds to a strain stabilized in the host animal, that is to say which exhibits stable characteristics in this host animal after several passages and in particular the following characteristics: identical period before the appearance of the disease and identical lesional profile during passages in all animals (degree of vacuolation of various parts of the brain); it corresponds to any strain stabilized under the abovementioned conditions, inducing a premature accumulation of PrPres in the spleen of the host animal, such as scrapie strains or bovine encephalopathy strains, in particular the scrapie strains called Chandler, ME7, 139A (M. E. Bruce et al., Scrapie strain variation and its implication in
Current topics in Microbiology and Immunology: Transmissible Spongiform Encephalopathies, Scrapie, BSE and related disorders,
1991, 172, 125-138), C506M3 (C. I. Lasmézas et al., J. Gen. Virol., 1996, 77, 1601-1609) or 263K (R. H. Kimberlin et al., J. Gen. Virol., 1977, 34, 295-304 and 1978, 39, 487-496) or the BSE strains called 4PB1 (C. I. Lasmézas et al., 1996, cited above) and 301V (C. F. Farquhar et al., J. Gen. Virol., 1996, 77, 1941-1946);
the said NCTA is preferably administered in a buffer suited to the route of administration selected in the form either of a crude tissue, preferably brain, homogenate, or of a PrPres pellet, obtained by appropriate centrifugation, from a crude tissue, preferably brain, homogenate;
the said NCTA may be administered by any route (oral route, parente
Beringue Vincent
Deslys Jean-Philippe
Lasmezas Corinne
Commissariat a l'Energie Atomique
Morgan & Lewis & Bockius, LLP
Parkin Jeffrey S.
LandOfFree
Method for screening substances with therapeutic action in... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for screening substances with therapeutic action in..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for screening substances with therapeutic action in... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3315568