Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Hormones – e.g. – prolactin – thymosin – growth factors – etc.
Reexamination Certificate
1998-01-22
2001-09-25
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Hormones, e.g., prolactin, thymosin, growth factors, etc.
C530S324000, C514S012200
Reexamination Certificate
active
06294656
ABSTRACT:
The invention relates to crystals and slow release formulations of Transforming Growth Factor-&bgr; (TGF-&bgr;).
BACKGROUND OF THE INVENTION
TGF-&bgr; plays a central role in many biological regulation pathways such as embryonal development or regeneration of tissue. It is a very potent biological agent which can be used also therapeutically for a series of different purposes.
TGF-&bgr; was originally purified to homogeneity from human platelets, human placenta and bovine kidney and identified as a homodimeric protein with a molecular mass of about 25.000 Da. First characterized by its ability to act synergistically with EGF or TGF-&agr; to induce anchorage-independent growth of untransformed NRK cells, recently, TGF-&bgr; has been shown to exhibit numerous regulatory effects on a wide variety of both normal and neoplastic cells indicating the importance of this protein as a multifunctional regulator of cellular activity. Depending upon the cell or tissue type, and the presence or absence of other growth factors, TGF-&bgr; may either stimulate mitogenesis, cell proliferation and growth, or may effectively inhibit said processes, or may exhibit other actions like e.g. control of adipogenesis, myogenesis, chondrogenesis, osteogenesis and immune cell function, stimulation of chemotaxis, or induction or inhibition of differentiation. Many of the actions of TGF-&bgr; are related to the response of cells or tissues to stress or injury, and to the repair of resultant damage. After inflammation, TGF-&bgr; plays the major role in the formation of granulation tissue, increases the expression of genes associated with extracellular matrix formation such as fibronectin, collagen and several protease inhibitors and stimulates collagen-matrix contraction by fibroblasts, suggesting its possible role in connective tissue contraction.
The term TGF-&bgr; represents a family of functionally and structurally closely related proteins. Until now, five distinct homodimeric TGF-&bgr;s designated as TGF-&bgr;1, TGF-&bgr;2, TGF-&bgr;3, TGF-&bgr;4 and TGF-&bgr;5 are described.
All TGF-&bgr;s are synthesized as 390 to 412 amino acid precursors that undergo proteolytic cleavage to produce the mature forms, which consist of the C-terminal 112 amino acids. In their mature, biologically active forms, TGF-&bgr;1 to 5 are acid- and heat-stable disulfide-linked homodimers of two polypeptide chains of 112 amino acids each. The complete amino acid sequences of human (Derynck, R. et al. (1985) Nature 316, 701-705), murine (Derynck, R. et al. (1986) J. Biol. Chem. 261, 4377-4379) and simian TGF-&bgr;1 (Sharples, K. et al. (1987) DNA 6, 239-244) show remarkable sequence conservation, differing only in a single amino acid residue. Comparison of the amino acid sequence of human TGF-&bgr;1, human TGF-&bgr;2 (deMartin, R. et al. (1987) EMBO J. 6, 3673-3677; Marquardt, H. et al. (1987) J. Biol. Chem. 262, 12127-12131) and human TGF-&bgr;3 (Ten Dijke, P. et al. (1988) PNAS 85, 4715-4719) has demonstrated that the three proteins exhibit in their mature forms about 70-80% sequence identity. A heterodimeric TGF-&bgr;1.2 has been isolated from porcine platelets and consists of one subunit of TGF-&bgr;1 disulfide-linked to one subunit of TGF-&bgr;2 (Cheifetz, S. et al. (1987) Cell 48, 409-415).
Recently, attempts have been undertaken aiming to produce TGF-&bgr;s by means of recombinant techniques rather than isolating these factors from natural sources (e.g. platelets) in order to obtain sufficient amounts for testing in various therapeutic modalities. However, it has proven to be extremely difficult to obtain biologically active recombinant TGF-&bgr;. As can be seen from the sequences depicted in the sequence listing under SEQ ID NOs.1 to 6, the 112 amino acids long mature forms of TGF-&bgr;1, TGF-&bgr;2 and TGF-&bgr;3 contain 9 cysteine residues. It has been shown for TGF-&bgr;2 that the 9 cysteine residues are forming 4 intrachain and 1 interchain disulfide bonds and that it has a complicated core structure (“disulfite knot”) [Schlunegger, M. P. and Gruetter, M. G., Nature 358:430-434(1992)]. It is known today that disulfide formation in TGF-&bgr;3 is analogous to TGF-&bgr;2 and that TGF-&bgr;3 exhibits the same complicated core structure.
Although expression of recombinant TGF-&bgr;s can be achieved in eukaryotic systems, the yields of biologically active, correctly folded material obtained are still far from being satisfactory. Therefore, attempts were made to produce biologically active TGF-&bgr; in a microbial host. However, in e.g. bacteria the intracellular conditions are not conducive to correct folding, disulfide bond formation and disulfide-stabilized dimerization which is apparently essential for activity. Thus, only very little biologically active TGF-&bgr; could be obtained after expression of the respective gene in
E. coli
under the control of the lambda promoter as described in European Patent Application EP-A-0 268 561. Another report describes the expression of a TGF-&bgr; cDNA in
E. coli
under the control of the trp promoter yielding a radioactively labelled protein band with an apparent molecular weight of 13'000 Da in an autoradiogram of a SDS polyacrylamide gel, but no activity was measured (Urushizaki, Y. et al. (1987) Tumor Res. 22, 41-55). However, in e.g. the European patent application EP-A-0 433 225 a successful process for the production of biologically active, dimeric TGF-&bgr;-like protein is described, in which a mild detergent is used for the folding of TGF-&bgr; isolated from inclusion bodies.
However, in preparing a pharmaceutically acceptable formulation the problem arises that TGF-&bgr;3 tends to absorb unspecificaly to several materials and is therefore difficult to administer in exact quantities. Moreover, like all proteins, TGF-&bgr;3 is sensitive towards oxidative agents such as oxygen in air.
It was found in the present invention that the use of TGF-&bgr;3 crystals instead of the soluted form prevents both against instability and adsorption.
In Schlunegger at al., FEBS Lett.303:91-93(1992) crystallization of TGF-&bgr;2 is described by the hanging drop method described in Schär et al., J. Biol. Chem. 262:13724(1987) from a 25-35% polyethylene glycol 400 solution. Likewise, in U.S. Pat. No. 5,322,933 TGF-&bgr;1 and TGF-&bgr;2 crystals were prepared by the hanging drop method using 20% PEG 200 and 600, respectively.
OBJECT OF THE INVENTION
It is an object of the invention to provide TGF-&bgr;3 in a crystalline form in order to provide a form which shows no adsorption or less adsorption to the wall of vials than the soluted TGF-&bgr;3. It is further an object of the invention to provide TGF-&bgr; in crystalline form in order to provide a form which is more stable towards oxidative agents than the soluted form.
Another object of the invention is to provide TGF-&bgr;3 crystals which can be used for structure determination and for drug design.
A further object of the invention is to provide a pharmaceutical preparation of TGF-&bgr; which contains TGF-&bgr; crystals and can be used as or for the production of a slow release pharmaceutical preparation.
SUMMARY OF THE INVENTION
Surprisingly, it was found that TGF-&bgr;3 forms crystals which are stable in aqueous solutions. TGF-&bgr;3 crystals do not stick to the walls of the glass vessel and the protein is protected against degradation, and which can be used for structure determination and for drug design.
Moreover, it was surprisingly found that TGF-&bgr; crystals can be used as or for the preparation of slow release formulations for therapeutic applications of TGF-&bgr;. If crystalline TGF&bgr; is applied to the patient, the concentration of soluble and, thus, available TGF-&bgr; can be varied by selecting crystals with different solution properties or by the appropriate crystal size.
DETAILED DESCRIPTION OF THE INVENTION
The present invention concerns crystals of TGF-&bgr;3, preferably TGF-&bgr;3 crystals selected from the group consisting of crystals belonging to a hexagonal or trigonal space group, more preferably crysta
Arvinte Tudor
Grutter Markus
Mittl Peer
Low Christopher S. F.
Lukton David
Novartis Corporation
Pfeiffer Hesna J.
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