Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Stablizing an enzyme by forming a mixture – an adduct or a...
Reexamination Certificate
1996-12-03
2002-05-07
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Stablizing an enzyme by forming a mixture, an adduct or a...
C435S196000
Reexamination Certificate
active
06383788
ABSTRACT:
FIELD OF THE INVENTION
The present invention is related to results obtained from research on the formulation of deoxyribonuclease, otherwise referred to as DNase, a phosphodiesterase that is capable of hydrolyzing polydeoxyribonucleic acid (DNA).
The present invention relates generally to the preparation of liquid solutions of DNase that are protected from thermally induced aggregation of the DNase active principal component. The present invention relates additionally generally to the preparation of liquid solutions of DNase that are maintained stable at pHs of less than neutral.
It relates to these solutions per se and to their methods of preparation and to their use clinically or for preparing further formulations useful clinically in the treatment of disorders susceptible to the biological activity of DNase, as discussed in more detail infra.
BACKGROUND OF THE INVENTION
DNase is a phosphodiesterase capable of hydrolyzing polydeoxyribonucleic acid. DNase has been purified from various species to various degrees. The complete amino acid sequence for a mammalian DNase was first made available in 1973. See, e.g., Liao, et al.,
J. Biol. Chem.
248, 1489 (1973).
DNase has a number of known utilities and has been used for therapeutic purposes. Its principal therapeutic use has been to reduce the viscoelasticity of pulmonary secretions in such diseases as pneumonia and cystic fibrosis, thereby aiding in the clearing of respiratory airways. See, e.g., Lourenco, et al.,
Arch. Intern. Med.
142, 2299 (1982); Shak, et al.,
Proc. Nat. Acad. Sci.
87, 9188 (1990); and Hubbard, et al.,
New England Journal of Medicine
326, 812 (1992).
DNA encoding human DNase has been isolated and sequenced and that DNA has been expressed in recombinant mammalian host cells, thereby enabling the production of human DNase in mammalian commercially useful quantities. See, e.g., Shak, et al.,
Proc. Nat. Acad. Sci.
87, 9188 (1990). Recombinant human DNase (rhDNase) has been found to be useful clinically, especially in purified form such that the DNase is free from proteases and other proteins with which it is ordinarily associated in nature.
The means and methods by which human DNase can be obtained in pharmaceutically effective form is described in the patent applications cited above. Various specific methods for the purification of DNase are known in the art. See, e.g., Khouw, et al., U.S. Pat. No. 4,065,355, issued Dec. 27, 1977; Markey,
FEBS Letters
167, 155 (1984); and Nefsky, et al.,
Euro. Journ. Biochem.
179, 215 (1989).
The present application is predicated on the use of such DNase for formulation. DNase can be employed as such, as a mixture of deamidated and non-deamidated forms, or in isolated deamidated and non-deamidated forms. The preparation and separation of such forms are the subject matter of a patent application cited above.
The present invention is directed to the preparation of liquid solutions of DNase (including all of its biologically active forms as previously noted) that are stable to thermally induced aggregation of DNase.
The present invention is directed to the preparation of stabilized liquid solutions of DNase (including all of its biologically active forms as previously noted). These liquid solutions containing DNase in substantially non-deamidated form are maintained at pHs of less than neutral in stable form such that precipitation of material does not occur to any substantial extent, and therefore, the solutions are in a clear form suitable for pharmaceutical administration. Such less than neutral pH levels result in a reduction of the rate of deamidation of the DNase principle during storage. At storage at elevated temperatures (upwards of 37° C.), such lower pH solutions result in precipitation products. The present invention, in an aspect, stabilizes such solutions from such precipitation.
SUMMARY OF THE INVENTION
The present invention is predicated upon the finding of an exceptional characteristic found attributable to a particular component which in liquid solution together with DNase as biologically active principle, protects said DNase from thermally induced aggregation. This particular component in liquid solution together with DNase as biologically active principle protects and stabilizes the solution from precipitation effects resulting in clear solutions which are suitable for pharmaceutical administration.
The pharmaceutical specifications permitting the storage with suitable shelf life of DNase requires the retention of at least 80% biological potency at from 2 to 8° C. over a sustained period of time. The recommended pH of such solutions are about neutral, more particularly approximately 6.5. It has been discovered that at this pH range, deamidation occurs at a relatively constant rate resulting in solutions in which the DNase component is increasingly deamidated. The deamidation takes place at the asparagine residue that occurs at position 74 in the amino acid sequence of native mature DNase. Attention is directed to U.S. Ser. No. 07/895,300, filed Jun. 8, 1992. In that application attention is focused on the separation of deamidated and non-deamidated DNase from one another for separate formulation into pharmaceutically administrable forms.
In this aspect of the present invention, it has been found that lowering the pH of such liquid solutions containing DNase substantially reduces the rate constant of deamidation resulting in liquid formulations which are relatively stable as to deamidation and thus the DNase remains in its non-deamidated form which is biologically more potent. However, the reduction of pH results in a by-process of precipitation of materials from the liquid solutions when stored at about 37° C., which is unacceptable from a pharmaceutical formulation standpoint.
The present invention in this aspect relates to the stabilization of such less than neutral pH liquid solutions containing DNase from precipitation resulting in solutions that are pharmaceutically administrable.
The introduction and use of calcium ion (Ca
+
2) protects such less than neutral pH liquid solutions of DNase from precipitation resulting in clear solutions that are suitable for pharmaceutical storage and administration, without the need for refrigeration. Thus, the present invention in this aspect relates to a method of stabilizing liquid solutions containing DNase as active principle, such solutions being at a pH of less than neutral such that deamidation is deterred or inhibited which comprises employing amounts of calcium ion in said solution that protect such solutions from precipitation resulting in liquid formulations of DNase that are suitable for storage and ultimate pharmaceutical administration. These solutions are maintained in a stable form without any or any substantial precipitation that otherwise occurs at pHs of less than neutral. Further, these solutions result in a minimal deamidation of the active component of DNase.
In the alternative aspect of the present invention, in signal distinction from certain other divalent cations, calcium ion (Ca
+2
) additionally protects DNase from thermally induced aggregation in liquid solution. Thus, the present invention relates to a process of minimizing thermally induced aggregation of DNase in liquid solution comprising DNase as active principle which comprises employing DNase-aggregation-minimizing amounts of calcium ion in said solution.
In a further embodiment of this alternative aspect of this invention, protection of DNase from thermally induced aggregation in liquid solution is obtained by the addition of sugars to said solutions, either in lieu of or additional to the presence of calcium ion.
Thus, the present invention is directed to methods for the preparation of liquid solutions comprising DNase as active principle which comprising using in said solutions an amount of calcium ion and/or sugar that: 1) minimizes DNase aggregation brought about from thermal instability (a DNase-aggregation-minimizing amount) and an amount of calcium ion that 2) stabilizes said solutions that are at a pH o
Chan Hak-Kim
Gonda Igor
Shire Steven J.
Weck Suzanne Sin-Mui Lo
Flehr Hohbach Test Albritton & Herbert LLP
Genentech Inc.
Weber Jon P.
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