Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Genetically modified micro-organism – cell – or virus
Reexamination Certificate
2001-11-20
2003-06-24
Nguyen, Dave T. (Department: 1633)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Genetically modified micro-organism, cell, or virus
C514S04400A, C435S320100, C435S091400
Reexamination Certificate
active
06582694
ABSTRACT:
The present invention relates to a method for preparing an aerosol comprising a virus.
Aerosol formulation of various substances, in particular medicinal substances, has been known for a very long time. Stability and negligible sedimentation rate constitute the essential features of an aerosol. For this reason, on the one hand, in order to prevent the particles aggregating with one another or displaying too high a sedimentation rate, properties incompatible with good efficiency, the proportions of constituents must satisfy a number of specific constraints due, in particular, to different environmental parameters such as the degree of humidity and the temperature, and on the other hand the formulation must take account of a number of difficulties mainly associated with the efficiency of the defence of the respiratory tract against aerial contaminants (clearance mechanisms), which tends to degrade inhaled active principles rapidly.
In particular, in the case where the active principle consists of viruses, the integrity of the viral particle is necessary for infection and penetration of the cells of the pulmonary epithelium, thereby imposing very specific conditions for obtaining the aerosol.
Generally speaking, during an aerolization, a virus may be inactivated as a result of three major causes. In the first place, loss of infectious power may result from the damage undergone by the virus during the nebulization process (spraying into the airways), it may also be inactivated in the aerosol by dehydration, and lastly it may be degraded in the potentially hostile environment represented by the mucus which coats the whole of the respiratory tract and which contains a large number of (proteolytic and other) enzymes.
It has now been demonstrated that viral inactivation can be limited and maintained within reasonable limits by applying, in order to obtain the aerosol, the method according to the invention employing particular conditions of nebulization and of packaging. In addition, this method enables the virus to be delivered effectively and in suitable amounts to the lung, in particular in the tracheobronchial passage.
Accordingly, the subject of the present invention is a method for obtaining a viral aerosol permitting, in particular, its administration via the airways of a mammal, characterized in that:
(a) a dilute viral suspension is prepared corresponding to the dilution of a viral suspension containing 10
4
to 10
13
plaque forming units (pfu) of a virus in an aqueous solution containing at least 6 to 12 g/l of a salt of a monovalent cation or 50 to 100 g/l of a hexose; and
(b) the dilute viral suspension is nebulized with a gas pressure of 0.5 to 3.5 bars or an ultrasound frequency of 2 to 5 MHz.
The method according to the present invention is applicable to a very large number of viruses which can be administered by aerosol, in particular viruses chosen from the group consisting of poxviruses, retroviruses, herpesviruses, adeno-associated viruses, rhinoviruses, influenza viruses and adenoviruses.
In the context of the present invention, the term “virus” denotes both a natural virus as found in nature, and a modified virus whose genome contains modifications relative to that of the parent virus from which it originates. It can be an attenuated virus which has lost all or part of its pathogenic power relative to the natural virus from which it is derived. Its genome is modified in vivo in the course of successive passages in cell culture or in a living organism.
The term “virus” can also refer to a recombinant virus whose genome is modified in vitro by genetic engineering techniques. The modification can, for example, enable at least one gene essential to the viral replication to be inactivated (rendering the virus defective for replication), and/or a DNA fragment coding for a heterologous protein (normally not encoded by the natural virus) to be inserted. Insertion takes place in a suitable region of the viral genome, so as to permit the expression of the heterologous DNA fragment in a host cell. A host cell consists of any eukaryotic cell which can be infected by said virus, advantageously a human cell and preferably an epithelial cell of the tracheobronchial passage.
For the purposes of the present invention, the heterologous DNA fragment can originate from a eukaryotic organism or from a virus other than the one into which it is inserted. It may be isolated by any conventional technique in the field of the art, for example by cloning, PCR (Polymerase Chain Reaction) or chemical synthesis. It can be a fragment of genomic type DNA (containing all or part of the set of introns of the natural gene), of the complementary DNA type (cDNA; lacking introns) or of the minigene, that is to say mixed, type (containing at least all or part of an intron).
In accordance with the objectives pursued by the present invention, the heterologous DNA fragment may be placed under the control of the elements needed for its expression. “Elements needed” denotes the set of elements permitting transcription of said DNA fragment into messenger RNA (mRNA) or translation of the mRNA into protein.
The heterologous DNA fragment can code for (i) an intracellular protein, (ii) a membrane protein present at the surface of the host cell, or (iii) a protein secreted into the external medium. It can hence contain a signal sequence permitting secretion of the protein towards the membrane or out of the host cell.
Many recombinant viruses capable of benefitting from an administration by aerosol are described in the prior art. Their construction and propagation are within the capacity of a person skilled in the art. As examples, there may be mentioned Ad-&agr;-1AT (Rosenfeld et al., 1991, Science, 252, 431-434), into the genome of which the human gene coding for &agr;1-antitrypsin (&agr;1AT) is inserted, and Ad-CFTR (Rosenfeld et al., 1992, Cell, 68, 143-155), into the genome of which the human gene coding for the CFTR (for Cystic Fibrosis Transmembrane Conductance Regulator, in English) protein is inserted.
The method according to the present invention involves the preparation of a dilute viral suspension. This dilute viral suspension corresponds to the dilution of a viral suspension containing 10
4
to 10
13
pfu of virus in an aqueous solution containing at least 6 to 12 g/l of a salt of a monovalent cation or 50 to 100 g/l of a hexose.
In this step, the suspension to be diluted is generally composed of viruses placed in a buffered medium optionally containing a bivalent cation such as magnesium, calcium or manganese. This type of suspension also being usable for storage. For storage in frozen form, the suspension should be supplemented with a stabilizing agent such as glycerol at a concentration of at least 10%, or sucrose at a concentration of approximately 1 M.
The suspension of viral particles to be diluted can optionally comprise other substances, in particular human serum albumin (HSA), urea, sodium glutamate, glycine and inositol.
According to the method of the present invention, it is necessary for the viral suspension to comprise 10
4
to 10
13
, advantageously 10
6
to 10
12
and preferably 10
8
to 10
11
pfu of a virus; the activity of this suspension will be able to depend, in particular, on the virus used.
This suspension to be diluted is then diluted with the aqueous solution according to a suspension/aqueous solution ratio by volume of 1:5 to 1:20, advantageously of 1:10 to 1:20, preferably of 1:12 to 1:18, and as an absolute preference approximately 1:16.
The aqueous solution preferably comprises 6 to 12 g/l of a salt of a monovalent cation, preferably a sodium salt or a potassium salt, and as an absolute preference potassium chloride, sodium lactate and/or sodium chloride. The concentration of salt of a monovalent cation is preferably 6 to 10 g/l, and as an absolute preference approximately 9 g/l.
When the aqueous solution comprises 50 to 100 g/l of a hexose, and preferably approximately 50 g/l, possible hexoses are, in particular, glucose and mannose.
In addition, the aqueous solution can
Lamy Didier
Sene Claude
Burns Doane Swecker & Mathis L.L.P.
Nguyen Dave T.
Transgene S.A.
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