Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1999-08-10
2003-03-18
Ketter, James (Department: 1636)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C435S006120, C435S320100, C435S458000, C435S283100
Reexamination Certificate
active
06534483
ABSTRACT:
INTRODUCTION
The present invention relates to products and methods useful for the production of a single-vial lyophilized nucleic acid/formulating agent complex with favorable physical and dosage characteristics.
BACKGROUND OF THE INVENTION
None of the information provided herein is admitted to be prior art to the present invention, but is provided only to aid the understanding of the reader.
Gene therapy has become a major area of research in rug development. More practical and effective gene delivery methods continue to aid the advancement of the clinical and/or commercial uses of gene therapy, which generally are expected to deliver the product to the targeted cells in sufficient quantities.
In the past, incorporation of nucleic acid into a conventional dosage form has been a challenge due to the chemical degradation or physical aggregation of the nucleic acid prior to administration. The preferred method of administration of a nucleic acid complex is in a liquid form, so past methods have included storing the nucleic acid in a separate vial and combining the necessary components shortly prior to administration. This was a generally effective method, however it was relatively expensive and difficult to prepare and produced a rather unstable product which was somewhat difficult to administer. The stabilization of polynucleotide complexes by adding a cryoprotectant compound and lyophilizing the resulting formulation is described in Szoka et al., International Patent Publication No. WO 96/41873, published Dec. 27, 1996, entitled “Dry Powder Formulations of Polynucleotide Complexes”, incorporated herein by reference in its entirety, including any drawings.
In prior attempts to make nucleic acid formulations, the method of mixing has typically been a conventional, slow (but not controlled) mixing of the DNA complex and formulating agent. The result was a non-homogenous complex with particles of relatively large size (approximately 150 nanometers). For ease of administration of the DNA complex, it would be desirable to have a smaller and more uniform particle size. Thus, despite the above, there remains a need for a single-vial homogenized nucleic acid formulation of a relatively small and uniform particle size, especially one protected from degradation and with an increased ability to transfect cells relative to the non-formulated nucleic acid, as well as easier methods of preparation and storage.
SUMMARY OF THE INVENTION.
This invention features compositions and methods for a cost-effective production of isolated, enriched or purified nucleic acids, preferably DNA plasmid, in a conventional dosage form without significant reduction of its biological activity. Thus, the present invention provides a single-vial, homogenized complex created by in-line mixing, where the resulting complex is lyophilized for storage and re-hydrated for administration. The in-line mixing preferably utilizes two tubes which lead into a mixer and which will serve to produce a mixture of a nucleic acid and a formulating agent. The complex in this invention, though formulated in a single vial, maintains favorable physical characteristics and potency. The use of this single vial formulation will result in: (1) reduced manufacturing costs, (2) ease of manufacturing and quality control testing, (3) product stability, and (4) increased doctor/patient compliance and relative ease of administration. These attributes, and the details that follow, provide advantages over the previously used formulations.
Thus, in one aspect, the invention features an in-line mixer containing a confined flowing system of isolated, enriched or purified liquid nucleic acid molecules.
By an “in-line mixer” is meant a device through which liquids to be contacted with one another are passed and which is used for continuous or semibatch operations. The mixer may include tubing and together with the liquid forms a confined flowing system. The volume of liquid in the mixer is limited only by the size and shape of the mixer. The mixers may utilize mechanical agitation, but when mechanical agitation is not used, other methods such as jet mixers, injectors, orifices, mixing nozzles, valves, and pumps may be used. Many in-line mixers are used commercially in chemical and chemical engineering applications, such as in the treatment of petroleum distillates, in vegetable oil refining, in some metal extractions and other applications. Because many types of these mixers are commercially available for use in chemistry and chemical engineering, those skilled in the art could easily design and make other in-line mixers with minor modifications that would still be suitable for treating nucleic acids as described herein.
The in-line mixer preferably is made up of two inlets in a Y-shaped configuration which join at an intersection to form a single outlet. In one embodiment of the invention, the in-line mixer includes a static mixer after the Y-shaped intersection.
By “nucleic acid molecules”, it is meant polynucleotides, i.e., a polymer of deoxyribonucleotides (DNA) or ribonucleotides (RNA) which includes naked DNA, a nucleic acid cassette, naked RNA, nucleic acid contained in vectors or viruses, both RNA and DNA including: cDNA, genomic DNA, plasmid DNA or condensed nucleic acid, nucleic acid formulated with cationic lipids, nucleic acid formulated with peptides, antisense molecules, cationic substances, RNA or mRNA. Examples of suitable nucleic acid molecules include those described in Szoka et al., International Application “Dry Powder Formulations of Polynucleotide Complexes”, International Patent Publication No. WO 96/41873, published Dec. 27, 1996, and Rolland, et al., International Patent Publication WO 96/21470, published Jul. 18, 1996, entitled, “Formulated Nucleic Acid Compositions and Methods of Administering the Same for Gene Therapy” which are incorporated herein by reference in their entirety, including any drawings. These are only examples and are not meant to be limiting. Additionally the nucleic acid molecules may be one or more plasmids with a eukaryotic promoter that expresses one or more therapeutic molecules. The nucleic acid molecules are, in certain aspects and embodiments, isolated, purified or enriched, as defined below in the Detailed Description at Section I.D.
The nucleic acid molecule may be isolated from a natural source by cDNA cloning or subtractive hybridization or synthesized manually. The nucleic acid molecule may be synthesized manually by the triester synthetic method or by using an automated DNA synthesizer.
The term “cDNA cloning” refers to hybridizing a small nucleic acid molecule, a probe, to genomic cDNA. The probe hybridizes (binds) to complementary sequences of cDNA.
The term “complementary” describes two nucleotides that can form multiple favorable interactions with one another. For example, adenine is complementary to thymine as they can form two hydrogen bonds. Similarly, guanine and cytosine are complementary since they can form three hydrogen bonds. Thus if a nucleic acid sequence contains the following sequence of bases, thymine, adenine, guanine and cytosine, a “complement” of this nucleic acid molecule would be a molecule containing adenine in the place of thymine, thymine in the place of adenine, cytosine in the place of guanine, and guanine in the place of cytosine. Because the complement can contain a nucleic acid sequence that forms optimal interactions with the parent nucleic acid molecule, such a complement can bind with high affinity to its parent molecule.
In a preferred embodiment, the nucleic acid administered is plasmid DNA which includes a “vector”. The nucleic acid can be, but is not limited to, a plasmid DNA vector with a eukaryotic promoter which expresses a protein with potential therapeutic action, such as, for example; hGH, VEGF, EPO, IGF-1, TPO, Factor IX, IFN-&agr;, IFN-&bgr;, IL-2, IL-12, or the like.
As used herein, the term “plasmid” refers to a construct made up of genetic material (i.e., nucleic acids). It includes genetic elements arranged such that an inserted coding sequen
Bruno Maria
Lawson Luke
Logan Mark J.
Mumper Russ
Tagliaferri Jenna
Ketter James
Perkins Coie LLP
Valentis, Inc.
Wise Michael J.
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