Drug – bio-affecting and body treating compositions – Preparations characterized by special physical form – Particulate form
Reexamination Certificate
2003-04-17
2004-11-09
Page, Thurman K. (Department: 1615)
Drug, bio-affecting and body treating compositions
Preparations characterized by special physical form
Particulate form
C424S400000, C424S489000
Reexamination Certificate
active
06814982
ABSTRACT:
The present invention concerns a suspension of crystallized particles of an EPI-hNE protein, methods for preparing said suspension, a dry powder aerosol derived from said suspension, an inhalable pharmaceutical formulation comprising said suspension or said dry powder aerosol, and the use of said inhalable pharmaceutical formulation in the treatment of various pathological conditions.
International Patent Application WO 96/20278 to Ley et al. describes a number of genetically engineered novel proteins which inhibit human neutrophil elastase (hNE). As indicated in the above-cited patent application, human neutrophil elastase (also known as human leukocyte elastase) is one of the major neutral proteases of the azurophil granules of polymorphonuclear leukocytes. This enzyme is involved in the elimination of pathogens, and in connective tissue restructuring.
The principal systemic inhibitor of hNE is the &agr;-1-protease inhibitor, formerly known as &agr;1 antitrypsin. In a certain number of pathological situations (hereditary disorders, chronic bronchitis, emphysema, cystic fibrosis), this inhibitor is either not present in sufficient amounts in the bloodstream or is inactivated, leading to uncontrolled elastolytic activity of hNE, which causes extensive destruction of lung tissue.
WO 96/20278 thus proposes novel proteins which are stable, non-toxic, highly efficacious inhibitors of hNE. These inhibitors form part of a group of inhibitors derived from a Kunitz-type inhibitory domain found in basic pancreatic trypsin inhibitor (BPTI) or a protein of human origin, namely the light chain of human Inter-&agr;-trypsin inhibitor (ITI). They are, inter alia, EPI-hNE-1, EPI-hNE-2, EPI-hNE-3 and EPI-hNE-4. The inhibitors of WO 96/20278 are produced by biotechnological methods and contain modified DNA sequences, with respect to the biological Kunitz domains, which render them highly potent. One of these inhibitors, EPI-hNE-4, is of particular interest.
WO 96/20278 describes preparation of
Pichia pastoris
production systems for hNE inhibitors EPI-hNE1, EPI-hNE-2, EPI-hNE-3 and EPI-hNE-4, protein production and purification (see in particular Examples 10-15).
Yeast
Pichia pastoris
mutant strain GS115 containing a non functional histidinol dehydrogenase gene (his4) was transformed by expression plasmids comprising a sequence encoding the
S. cerevisiae
mating factor alpha prepro peptide fused directly to the amino terminus of the desired hNE inhibitor, under control of the upstream inducible
P. pastoris
aox1 gene promoter and the downstream aox1 transcription termination and polyadenylation sequences. The expression plasmids were linearized by SacI digestion and the linear DNA was incorporated by homologous recombination into the genome of the
P. pastoris
strain GS115 by spheroplast transformation, selection for growth in the absence of added histidine and screening for methanol utilization phenotype, secretion levels and gene dose (estimated by Southern Blot). Strains estimated to have about four copies of the expression plasmid integrated as a tandem array into the aox1 gene locus were thus selected.
Cultures of selected strains were first grown in batch mode with glycerol as the carbon source, then, following exhaustion of glycerol, grown in glycerol-limited feed mode to further increase cell mass and derepress the aox1 promoter and finally in methanol-limited feed mode. During the latter phase the aox1 promoter is fully active and the protein is secreted into the culture medium.
The EPI-hNE protein is then purified. The specific purification procedure varies with the specific properties of each protein. Briefly, the culture medium is centrifuged, the supernatant is subjected to microfiltration and subsequently to ultrafiltration, optionally to diafiltration, and then the protein is recovered by ammonium sulfate precipitation and ion exchange chromatography.
European Patent Application No. 00203049.2 and international patent application PCT/FR 01/02699 claiming priority thereof, filed by the applicant company, describe an improved process for the purification of an EPI-HNE protein of pharmaceutical quality, from the culture medium of a host strain for the expression of said proteins, comprising the steps of:
(a) passing a derived part of the culture medium over an expanded, bed of cationic exchange adsorbent in order to recover an eluate,
(b) conducting separation of proteins, according to their hydrophobicity, on the resulting eluate,
(c) passing the resulting eluate over a cationic exchange column,
(d) optionally filtering the resulting medium under sterile conditions, and
(e) optionally lyophilising the resulting filtrate in order to recover an EPI-HNE protein.
The solution obtained at the end of step (d) or a freeze dried powder obtained therefrom can be used to prepare the suspension according to the present invention, said suspension being capable of being incorporated in an inhalable pharmaceutical formulation according to the invention.
The applicant company, having perfected a purification method of EPI-hNE proteins, particularly EPI-hNE-4, has subsequently devoted a great deal of research and effort to the development of pharmaceutical compositions containing the purified EPI-hNE proteins.
In fact, the Applicant Company has concentrated on the development of a buccal inhalable pharmaceutical composition containing a solution of Epi-hNE-4. However, in the course of product development, it was found that the solution of EPI-hNE-4, once in the nebulizer, was unstable and tended to precipitate, rendering the solution turbid.
It was first thought that the precipitated form of the protein would be therapeutically inactive. However, in a surprising and unexpected manner, it was found that the precipitated form was a crystallized form of EPI-HNE4 and that this crystallization did not adversely affect the activity of the protein. The term “crystallized form of EPI-hNE4” here means an insoluble form of this protein, having a rod-like structure and a particle size mainly below 10 &mgr;m.
The applicant company thus turned its efforts towards developing a suspension of the EPI-hNE proteins in which the protein would be in crystalline form, said suspension being capable of being incorporated into a pharmaceutical composition, in particular an inhalable pharmaceutical formulation.
It was surprisingly found that it was possible to prepare a suspension which is stable at room temperature under certain specific conditions of concentration and pH, thereby allowing the preparation of pharmaceutical compositions incorporating said suspension which are stable at room temperature. This room temperature stability is of particular importance for ambulatory treatments.
The use of a suspension of EPI-hNE proteins, instead of a solution, constitutes a major advantage in the preparation of inhalable pharmaceutical compositions, insofar as it allows the development of a formulation which is more concentrated in active substance, thereby permitting administration of the drug in a shorter time frame.
This is an important factor in the administration of inhalable drugs since the time period of inhalation required can often be long, which is perceived as a major constraint and may hence lead to poor patient compliance.
Thus, the present invention concerns a suspension of an EPI-hNE protein, said suspension being characterized in that the EPI-hNE protein is present in the form of crystalline particles mostly having a particle size comprised between 1 and 6 &mgr;m, in particular between 3 and 6 &mgr;m, as determined by laser granulometry, the concentration of the suspension in the EPI-hNE protein being comprised between 1 and 80 mg/ml, preferably between 2 and 50 mg/ml, most preferably depending on the amount of the EPI-hNE protein required for the treated therapeutic condition, in an aqueous vehicle at a pH comprised between 3and 8, preferably 4 and 6, most preferably at a pH of 4.0 to 5.0.
The above suspension is stable as to its biological activity and particle size distribution for a period of at leas
Bokman Anne
Poncin Alain
Saudubray François
Debiopharm S.A.
Oh Simon J.
Page Thurman K.
Sturm & Fix LLP
LandOfFree
Suspension of an EPI-HNE protein, process of preparation... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Suspension of an EPI-HNE protein, process of preparation..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Suspension of an EPI-HNE protein, process of preparation... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3363067