Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1998-12-22
2002-05-14
Nguyen, Dave T. (Department: 1633)
Chemistry: molecular biology and microbiology
Vector, per se
C435S069100, C435S325000, C435S455000
Reexamination Certificate
active
06387695
ABSTRACT:
STATEMENT REGARDING FEDERALLY-SPONSORED R&D
Not applicable.
REFERENCE TO MICROFICHE APPENDIX
Not applicable.
FIELD OF THE INVENTION
The present invention relates to formulations of nucleic acid pharmaceutical products, specifically formulations of nucleic acid vaccine products and nucleic acid gene therapy products which comprise citrate and/or triethanolamine at pharmaceutically effective concentrations. DNA stability is increased in the pharmaceutical formulations of the present invention, allowing for long term storage and ease of distribution of DNA-based pharmaceutical products.
BACKGROUND OF THE INVENTION
DNA plasmid vaccines undergo a physiochemical change from supercoiled to the open circular and linear form during storage as a pharmaceutical entity. A variety of storage conditions such as low pH, high temperature and low ionic strength can accelerate this deleterious process.
The nucleic acid component of the formulations disclosed within this specification are free of nuclease activity, leaving the chain scission reaction as the sole known cause in converting supercoiled plasmid to the open circular and linear forms. The chain scission reaction takes on two distinct chemical mechanisms: (1) depurination followed by &bgr;-elimination and/or (2) free radical oxidation. Previous attempts to overcome these chemical-induced destabilizing mechanisms have included lyophilizing formulations containing DNA or by adding combinations of free radical scavengers and metal ion chelators to an aqueous formulation. In WO97/40839, the combination of EDTA and ethanol has been reported to be an effective inhibitor of free radical oxidation of DNA. However, this formulation requires the addition of two separate components to enhance stability. More importantly, the combination of EDTA and ethanol significantly increases the osmolarity of the formulation while the inclusion of EDTA in vaccine formulations has not of yet received worldwide acceptability. WO97/40839 also discloses various DNA vaccine formulations, but does not teach or suggest the use of citrate and/or triethanolamine to enhance DNA stability.
It would be useful to identify pharmaceutical formulations comprising a physiologically advantageous pH and osmolality which enhance DNA vaccine stability over longer periods of time at ambient temperatures. The present invention addresses and meets these needs by disclosing the ability to enhance stability of such formulations by the addition of citrate and/or triethanolamine at pharmaceutically acceptable concentrations.
SUMMARY OF THE INVENTION
The present invention relates to nucleic acid formulations of pharmaceutical products, especially nucleic acid formulations for use as nucleic acid vaccine products and nucleic acid gene therapy products. The formulations of the present invention stabilize the conformation of nucleic acids in pharmaceutical products. It will be evident upon review of this specification that the preferred template for stabilization is plasmid DNA. However, the formulations of the present invention may also be used to enhance stability of other nucleic acid molecules which are a part of a pharmaceutical formulation, including but not limited to other DNA forms such as genomic DNA molecules, complementary DNA (cDNA) molecules, which may be single (coding or noncoding strand) or double stranded, as well as synthetic DNA, such as a synthesized, single stranded polynucleotide. Additionally, nucleic acid pharmaceutical formulations of the present invention may also be used to enhance stability of other ribonucleic acid molecules (RNA), including but not limited to RNA-based vaccines, RNA-based gene therapy products, isolated messenger RNA (mRNA), isolated transfer RNA (tRNA) and synthetic RNA oligonucleotides.
The present invention relates to a nucleic acid pharmaceutical formulation which comprises citrate at a concentration which results in an increase in stability of the nucleic acid entity. It will be well within the purview of the skilled artisan to use the teachings of this specification to generate various nucleic acid formulations which contain citrate and which in turn, due to the presence of citrate, result in increased nucleic acid stability. To this end, it will be known that variations in formulation composition may include, but are not limited to, variations in pH within an acceptable range for storage of a biologically active nucleic acid template, preferably in a range from about 7.0 to about 9.5; variations in the buffers utilized, including but not limited to Tris, glycine, sodium phosphate, potassium phosphate, lithium phosphate, sodium bicarbonate, potassium bicarbonate, lithium bicarbonate, sodium borate, potassium borate, lithium borate and sodium, potassium or lithium citrate; variations in salt concentration; and addition of sugars including but not limited to 6-carbon polyhydric alcohols such as sorbitol, mannitol and dulcitol and/or disaccharides such as sucrose, lactose, maltose or trehalose, as well as various adjuvants which may stimulate the intended in vivo application. It will be evident to one of ordinary skill in the art that a formulation of the present invention will have a tonicity that is acceptable for parenteral injection.
One embodiment of the present invention relates to a nucleic acid formulation comprising a nucleic acid molecule, a pharmaceutically acceptable buffer to adjust the pH from about 7.0 to about 9.5, and citrate or a pharmaceutically acceptable salt thereof, including but not limited to sodium, potassium, lithium and triethanolamine salts.
Another embodiment of the present invention relates to a nucleic acid formulation wherein the nucleic acid template is a plasmid DNA construct and which comprises a pharmaceutically acceptable buffer at a pH from about 7.0 to about 9.5, and citrate or a pharmaceutically acceptable salt thereof, including but not limited to sodium, potassium, lithium and triethanolamine salts.
Another embodiment of the present invention relates to a nucleic acid formulation wherein the nucleic acid template is a plasmid DNA construct and which comprises a pharmaceutically acceptable buffer at a pH from about 7.7 to about 8.5, and citrate or a pharmaceutically acceptable salt thereof, including but not limited to sodium, potassium, lithium and triethanolamine salts.
An additional embodiment of the present invention relates to a nucleic acid formulation wherein citrate is a component of the formulation as well as a DNA plasmid template and phosphate buffered saline (PBS, contains 150 mM NaCl unless specified otherwise) wherein the pH is adjusted from about pH 7.2 to about pH 8.0 and the NaCl concentration is present in a range up to about 150 mM.
Yet another embodiment of the present invention relates to a nucleic acid formulation wherein citrate is a component of the formulation as well as a DNA plasmid template and 10 mM phosphate buffered saline, pH 7.7.
A preferred embodiment of the present invention relates to a nucleic acid formulation wherein citrate is present at about 100 mM in 10 mM phosphate buffer or is present from about 1 mM to about 20 mM in 10 mM phosphate buffered saline at pH 7.7.
An especially preferred embodiment of the present invention relates to a nucleic acid formulation wherein the DNA plasmid of interest is present in 10 mM citrate and 10 mM phosphate buffered saline at pH 7.7.
Another embodiment of the present invention relates to a nucleic acid formulation comprising a nucleic acid molecule, a pharmaceutically acceptable buffer to adjust the pH from about 7.0 to about 9.5, and triethanolamine or a pharmaceutically acceptable salt thereof. Triethanolamine may be added directly to the formulation as pure triethanolamine, which will form a salt in an aqueous solution. Triethanolamine may be added as a salt, the most common forms including but not being limited to the chloride, phosphate, bicarbonate, acetate, bromide, iodide, sulfate, succinate, citrate, malate and tartrate salts.
A further embodiment of the present invention relates to a nucleic acid formulation wher
Bruner Mark W.
Evans Robert K.
Volkin David B.
Xu Zheng
Hand J. Mark
Merck & Co. , Inc.
Nguyen Dave T.
Tribble Jack L.
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