Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Hormone or other secreted growth regulatory factor,...
Reexamination Certificate
2001-09-17
2003-11-18
Spector, Lorraine (Department: 1646)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Hormone or other secreted growth regulatory factor,...
C514S002600, C514S769000, C530S350000
Reexamination Certificate
active
06649168
ABSTRACT:
The present invention relates to pharmaceutical compositions containing TGF-&bgr;.
Transforming growth factor-type &bgr; (TGF-&bgr;) is a homodimeric protein with a molecular mass of about 25,000 D. It denotes a family of multifunctional cytokines which regulate cell proliferation and differentiation processes, activity in bone and connective tissue. Highest levels of TGF-&bgr; are found in blood platelets and bone. The pharmacological effects of TGF-&bgr; are acknowledged in the art which may be promotion and acceleration of wound healing, bone, cartilage, and tissue repair, the treatment of cancer, bone marrow protective agent, mediator of cardioprotectin, anti inflammatory or immunosuppressive agent, mediator of inductive tissue interactions, induction of angiogenesis, oral mucositis, or growth regulator in mammalian cell cultures.
“TGF-&bgr;” as used therein are members of the TGF-&bgr; family. Any of the TGF-&bgr; isoforms and related molecules may be used in the pharmaceutical compositions of this invention. Preferred TGF-&bgr; of the present invention are TGF-&bgr;1, TGF-&bgr;2 and BMPs such as BMP 2 and BMP 7, especially TGF-&bgr;3. TGF-&bgr;3 is a 25 kD homodimeric, disulfide-linked protein composed of two 112 amino acid polypeptides containing 9 cysteines each. The cysteines of the homodimeric protein form 4 intrachain disulfide bonds and one interchain disulfide bond.
“TGF-&bgr;” embraces TGF-&bgr; mutants, e.g. TGF-&bgr; proteins exhibiting similar-biological activities and differing from the native TGF-&bgr; proteins by simple or multiple mutations, e.g. replacement, addition or omission of amino-acids.
We have found one problem in developing a dosage form containing TGF-&bgr; because of its poor physical and chemical stability in aqueous solution and in powder form. The poor stability may be observed when TGF-&bgr; is analyzed by chromatographic methods, such as gel electrophoresis and HPLC.
We have further found that in solution TGF-&bgr; binds to the walls of the container which constitutes a further problem. Such adsorption phenomena are major obstacles in the development of stabile aqueous formulations such as prefilled gel bottles which are used more friendly for the patient and have reduced production costs. We have found for low therapeutical doses for TGF-&bgr; (e.g. below 10 &mgr;g/ml, e.g. between 0.001 and 10 &mgr;g/ml, e.g. between 1 and 10 &mgr;g/ml) the adsorption to surfaces has to be inhibited during the production of the dosage forms, during the storage and also before the use of the TGF-&bgr; formulation by the patient. The low concentrations used in formulations and the adsorption of TGF-&bgr; to surfaces impose also the development of highly sensitive analytical methods.
We have now found surprisingly that certain agents totally stop the adsorption of TGF-&bgr; to walls. Such agents may be used in different TGF-&bgr; dosage forms which have improved physical and chemical stability.
The present invention provides in one aspect a pharmaceutical composition comprising TGF-&bgr; and a water soluble salt chosen from calcium chloride, calcium phosphate, sodium acetate, potassium acetate, lithium acetate, ammonium acetate and ammonium bicarbonate, preferably calcium chloride and calcium phosphate.
The TGF-&bgr;s used in the water soluble salt pharmaceutical composition of the invention may be in the free form or in the form of their salts.
We have also developed an analytical method suitable for determining suitable concentrations of TGF-&bgr;, especially TGF-&bgr;3. When adsorption of a fluorescent molecule (protein) occurs to the cuvette walls the concentration of the molecule in the solution decreases. This change in concentration is monitored by changes (reduction) in the fluorescence intensity. With optimized slits in the excitation and emission monochromators the excitation beam is focused in a small region in the middle of the cuvette and the emission is collected also preferentially from the same region. This localized excitation and emission indicates that the contribution to the total emission intensity of chromophores which are bound to the cuvette walls to be minimal. In these conditions the fluorescence intensity is proportional to the concentration of the molecules in the aqueous solution (monitored in the middle of the cuvette). If binding occurs a decrease in the fluorescence intensity is observed. For TGF-&bgr; the intrinsic tryptophan fluorescence is used in the adsorption studies. To characterize an adsorption process the percentage from the total TGF-&bgr; in the cuvette which bound to the walls in a given time interval, e.g. 25 minutes, is measured. Fluorescence measurements are performed with a Spex Fluorolog® or a Spex Fluoromax® spectrophotometers with a stirred attachment in the cell holder at 25° C. Tryptophan fluorescence is excited at 280 nm and emission monitored at 340 nm. TGF-&bgr; is found to bind strongly to plastic, quartz and siliconised quartz cuvettes for a solution of TGF-&bgr; (1 &mgr;g/ml) in water. We have found a particularly good system for reproducibility of the majority of fluorescence adsorption studies if studies are performed in disposable plastic cuvettes (PMMA) supplied by Dispolob®, Kartell® P. N.1961.
A method to study protein adsorption to surfaces of different materials (other than the material from which the cuvettes are made) has also been developed. In these experiments TGF-&bgr; from a stock solution is added in a cuvette containing an aqueous solution (i.e. water, final TGF-&bgr; concentration 2 &mgr;g/ml) and the binding of TGF-&bgr; to the walls is monitored in time until the equilibrium state for the binding is reached (constant fluorescence intensity). Two sheets of the material needed to be studied are introduced in the cuvette and the changes in TGF-&bgr;3 fluorescence are monitored. If TGF-&bgr; is not binding to this material no change in time of the fluorescence intensity, i.e. no binding to the surface, is observed. If TGF-&bgr;3 binds to the new material a decrease in fluorescence intensity is measured.
In a preferred composition of the invention, the molar ratio of the water soluble salts ions to TGF-&bgr;, e.g. TGF-&bgr;3, is from 1:1 to 200:1, e.g. 10:1 to 100:1.
A man skilled in the art will appreciate that a wide variety of excipients may be used. For the water-soluble composition preferred components are e.g.:
a) a liquid solvent, e.g. an alcohol, e.g. ethanol or isopropanol,
b) a sugar or a sugar alcohol, e.g. mannitol, trehalose, sucrose, sorbitol, fructose, maltose, lactose or dextrans, preferably mannitol,
c) other excipients, e.g. polygeline, polysorbate 20, PVC Palinode® C, and/or methyl-cellulose 4000 cP
d) isotoning agents, e.g. sodium chloride,
e) an acid, e.g. citric acid monohydrate, acetic acid,
The amount of additives used can vary dependent on the intended use.
The water soluble pharmaceutical composition may comprise, e.g.
0.05 &mgr;g/ml to 100 mg/ml of TGF-&bgr;3, e.g. 0.1 &mgr;g/ml to 40 mg/ml
0.1 to 200 mg/ml of the salt, e.g. calcium chloride,
1 to 90 mg/ml of a liquid solvent, e.g. an alcohol
1 to 50 mg/ml of sugar or sugar alcohol,
0.5 to 20 mg/ml of an acidic compound, e.g. citric acid monohydrate or acetic acid.
Such solutions may be used for standard ampoules, vials, pre-filled syringes or multiple administration systems.
If desired, a freeze dried formulation which may be stable for long periods of time, e.g. 6 months at 40° C., without the need for refrigerated storage, may be obtained from a TGF-&bgr; solution.
The freeze dried product may be obtained in conventional manner from a suitable solution, e.g. the above-disclosed solution, e.g. having a pH of from 1 to 4.5, e.g. from 2.5 to 4. Preferably, the concentration of TGF-&bgr; in this solution before freeze drying is from 0.1 &mgr;g/ml to 40 mg/ml, e.g. 10 &mgr;g to 2 mg/ml.
The freeze dried product may be re-dissolved into a solution which may be stable for long periods, e.g. up to 1 week, e.g. at pH below pH 5, e.g. between pH 2.5 and pH 4. In such a solution TGF-&bgr; is in the range of from
Arvinte Tudor
Schote Uwe Thomas
Sigg Juergen
Andres Janet L.
Novartis AG
Spector Lorraine
Thallemer John D.
Wilusz E. Jay
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