Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1999-10-26
2001-10-16
LeGuyader, John L. (Department: 1635)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C424S450000, C424S489000, C424S490000, C435S458000, C435S459000
Reexamination Certificate
active
06303582
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to compositions and methods for delivering nucleic acids to the lungs of humans and other animal hosts. More particularly, the present invention relates to compositions which are formed by incorporating nucleic acid constructs within a hydrophilic excipient matrix. The resulting compositions are then stored and utilized in dry powder form.
A form of human gene therapy which is receiving increasing interest relies on the in vivo delivery of functional nucleic acids, usually structural genes, to certain target cells within a human or other host. The nucleic acids may be incorporated into carriers such as viruses, liposomes, or the like, and will be delivered under conditions which result in uptake of the genes into the target cells, with subsequent expression of the genes for an extended period of time.
Of particular interest to the present invention, it has been demonstrated that nucleic acid constructs can be delivered to the lungs of mice and rats by different routes, including intratracheal administration of a liquid suspension of the nucleic acids and inhalation of an aqueous aerosol mist produced by a liquid nebulizer. Although holding great promise, both methods for the delivery of nucleic acids to the lungs suffer from certain drawbacks. Intratracheal administration is not suitable for routine therapeutic use in humans and has a very low patient acceptability. Moreover, intratracheal instillation often results in very uneven distribution of a dispersion in the lungs, with some regions receiving very little or no material. The use of a liquid nebulizer enjoys higher patient acceptability and achieves better distribution, but requires time-consuming equipment set-up, can require prolonged periods of treatment to achieve an adequate dosage, can inactivate a viral carrier, and can result in undesirable aggregation or degradation of the nucleic acids within the aerosol mist. Aggregated nucleic acids will generally be less suitable for uptake into host target cells.
For these reasons, it would be desirable to provide improved compositions and methods for the aerosol delivery of nucleic acids. The compositions will preferably be in a dry powder form which can be readily dispersed in a flowing air stream to provide a dry aerosol for delivery to a patient. The dry powder formulations will permit delivery of required dosages of nucleic acids in a very rapid manner (typically in several or fewer breaths) and will be suitable for storage over extended periods. The dry powders are delivered to particular target regions within the host and are readily dispersed over the internal surfaces of lung, where the powder dissolves in the moist layer over the surfaces to thereby release nucleic acids to interact with the target cells.
2. Description of the Background Art
Stribling et al. (1992) J. B
IOPHARM.
S
CI.
3:255-263, describes the aerosol delivery of plasmids carrying a chloramphenicol acetyltransferase (CAT) reporter gene to mice. The plasmids were incorporated in DOTMA or cholesterol liposomes, and aqueous suspensions of the liposomes were nebulized into a small animal aerosol delivery chamber. Mice breathing the aerosol were found to at least transiently express CAT activity in their lung cells. Rosenfeld et al. (1991) S
CIENCE
252:431-434, describes the in vivo delivery of an &agr;1-antitrypsin gene to rats, with secretion of the gene product being observable for at least one week. An adenoviral vector containing the gene was diluted in saline and instilled directly into the rat trachea. Underwood et al. (1991) J. P
HARMACOL.
M
ETH.
26:203-210, describes the administration of dry powder bronchodilators in a lactose carrier to pig lungs. U.S. Pat. No. 5,049,388 describes the delivery of liquid aerosols containing liposomes to the lungs. Friedman (1989) S
CIENCE
244:1275-1281 is a review article describing human gene therapy strategies. In Felgner and Ringold (1989) N
ATURE
387-388, it is described that the presence of certain polyvalent ions can reduce transfection efficiency in vitro using liposomes. Gershon et al. (1993) B
IOCHEMISTRY
32:7143-7151 describe that multivalent anions such as citrate or phosphate can induce fusion of positively-charged liposomes used for transfection.
SUMMARY OF THE INVENTION
In one aspect, the present invention is directed to dry powder nucleic acid compositions comprising nucleic acid constructs (typically small particles) dispersed within a matrix of hydrophilic excipient material to form large aerosol particles. Usually, the nucleic acid particles will be present in excess powdered excipient material, usually being the same excipient which forms the matrix. The powdered aerosol particles will have an average particle size in the range from 0.5 &mgr;m to 200 &mgr;m, usually being in the range from 0.5 &mgr;m to 5 &mgr;m for lung delivery with larger sizes being useful for delivery to other moist target locations. The nucleic acid constructs may comprise bare nucleic acid molecules, viral vectors, associated viral particle vectors, nucleic acids present in a vesicle, or the like.
The dry powder nucleic acid compositions may be prepared by suspending the nucleic acid constructs in an aqueous solution of the hydrophilic excipient and drying the solution to produce a powder comprising particles of the nucleic acid construct dispersed within the dried excipient material, usually in the presence of excess powdered excipient. The weight ratio of nucleic acid construct to hydrophilic excipient in the initial solution is in the range from 2:1 to 1:100, preferably from 1:1 to 1:10, and the solution may be dried by spraying droplets into a flowing gas stream (spray drying) or by vacuum drying to produce a crude powder followed by grinding to produce a final powder.
In the case of particles intended for lung delivery, having a particle size from 0.5 &mgr;m to 5 &mgr;m, each particle may contain from 10 to 10
7
nucleic acid constructs, usually from 10
2
to 10
5
nucleic acid constructs, and preferably from 10
3
to 10
4
nucleic acid constructs. The constructs may be uniformly or non-uniformly dispersed in each particle, and the particles in turn will often be present in excess powdered excipient, usually at a weight ratio (nucleic acid construct:excipient powder free from nucleic acids) in the range from 1:1 to 1:10
3
, and more usually from 1:10 to 1:500.
In a preferred embodiment of the present invention, aqueous solutions will contain as the nucleic acid construct, nucleic acids in liposome vesicles. Preferably, such solutions will be substantially free from buffering agents and salts. It has been found that drying, particularly spray drying, of such buffer-free solutions results in powders having enhanced transfection activity compared to powders formed by drying the same liposome vesicles in buffered solutions. In contrast, aqueous solutions in which the nucleic acid constructs comprise viral vectors usually will be buffered to enhance stability of the viral vectors.
In a second preferred aspect of the present invention, the dry powder nucleic acid compositions will be prepared by spraying droplets of the liquid solution into a heated gas stream over a short time period, typically at temperatures ranging from 50° C. to 150° C. over a period from 10 msec to 100 msec, in a spray dryer. The resulting powder comprising particles containing nucleic acid constructs (and usually containing powdered excipient free from nucleic acids) will then be collected in a partially cooled environment, typically maintained at 5° C. to 50° C., and thereafter stored at a temperature from 5° C. to 25° C. at a low humidity, typically below 5% RH. It has been found that such collection and storage conditions help to preserve and stabilize the compositions and to enhance transfection efficiency.
Methods for delivering nucleic acid constructs according to the present invention comprise directing the dry powder containing the nucleic acid constructs to a moist target location in a
Eljamal Mohammed
Foster Linda
Patton John S.
Platz Robert M.
Cagan Felissa H.
Evans Susan T.
Hurst Stephen L.
Inhale Therapeutic Systems Inc.
Larson Thomas G
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