TB-500 Thymosin Beta-4 is a synthetic peptide of the naturally occurring healing protein present in about all human and mammalian cells known as Thymosin Beta 4 (TB4), which is a 36 amino acid sequence that is produced by and secreted by human fibroblasts as an inactive precursor polypeptide containing a 5 amino acid signal thymosin beta 4 half life sequence.
enhances the healing process of injured tissues of mammalian origin, such as epidermis, dermis, tendons, ligaments and synovium, but not other tissues such as muscle, bone and cartilage. TB-500 is non-toxic and immunologically non-active in in vitro, animal and human subjects. In-vitro studies show that TB-500 is also bioavailable and is not readily metabolized. In a preliminary study, TB-500 was found to penetrate into the intercellular matrix of the epidermis where the peptide was taken up by keratinocytes and dermal fibroblasts.
How Does TB-500 Thymosin Beta-4 Work?
However, the in vivo mechanism(s) of uptake and penetration of TB-500 into the skin are unknown. The hypothesis is that the major absorption site for TB-500 is in the skin. The long term objective is to develop an efficient means for administering TB-500 topically to a patient with extensive or deep tissue injury to enhance tissue repair and regeneration. This objective will be accomplished by demonstrating that TB-500 penetrates the deep tissues of the epidermis and the dermis, whereupon it enters both the epidermis and dermis cell population.
This will be accomplished by administering radiolabelled TB-500 to animals and evaluating the concentration of the radiolabelled peptide in various tissues by standard methods of radioautography and scintigraphy. TB-500 will be prepared in an efficient form such that it may be produced on a commercial scale. The specific aim of this proposal is to develop a formulation of TB-500 that will protect the peptide from enzymatic degradation. When ready, this formulation will be evaluated by the same methodology as described above.
If the TB-500, formulated and absorbed into the epidermis, is shown to enter into the dermis and reach the cell population, it will be assumed that TB-500 is absorbed into the deeper layers of the dermis. A second hypothesis is that TB-500 penetrates the deep dermis and reaches the intercellular matrix of the dermis, whereupon it is taken up by dermal fibroblasts, keratinocytes and endothelial cells. If this hypothesis is confirmed, TB-500 would be shown to enter the intercellular matrix, which contains an interconnected network of collagen fibers.
This study will establish an effective means of topical application of TB-500 to patients with skin wounds and other injuries. As an example of an applied clinical indication for TB-500, it would be possible to use the peptide in patients with severe burns that often progress to chronic ulcers. The peptide would be applied over the burns in topical form to prevent further injury, inhibit scar formation, and facilitate the wound healing process. These studies will involve animal models, and the experiments will also be performed in human subjects. The experimental data obtained will be utilized in the development of a large-scale preparation of the TB-500 peptide.
TB-500 is a synthetic peptide of the naturally occuring healing protein present in about all human and mammalian cells known as Thymosin Beta 4 (TB4). The peptide substance has demonstrated a significant growth stimulation of cultured T and B lymphocytes (Ludwig, A., et al., Immunol. Letters 31:81-86, 1994). It shows high antimicrobial activity against a broad spectrum of pathogens (Lalvani, M., et al., Chemotherapy 31:13-16, 1988), including several types of resistant Gram negative bacteria (Witt-Eggeling, M., et al., Antimicrob. Agents Chemother. 37:2189-95, 1993; St. Ritter, C., et al., Med. Microbiol. Immunol. 197:137-45, 1992), and good anti-tumour activity (Yap, A. Y., et al., Cancer Res. 48:6604-08, 1988). A recent study found that TB4 acts as an anti-angiogenesis agent, inhibiting vascular endothelial cell proliferation (Hofleitner, E. M., et al., J. Biol. Chem. 273:9238-47, 1998).
Amino acid composition of the polypeptide of TB4 is different from Thymosin alpha1 and Thymosin beta10. The Thymosin beta4 peptide contains two disulphide bridges, while thymosins alpha1 and beta10 do not have disulphide bridges. It has a higher molecular weight, is rich in polar amino acids, and contains a considerable amount of proline (Lilenius, J., et al., Tumor Biol. 12:249-54, 1991). The Thymosin beta4 peptide, having a high content of proline (50-60%) and histidine (20-25%), shows high proteolytic resistance (Saraste, J., et al., J. Pharm. Biomed. Anal. 12:567-72, 1990). In a recent study, a series of synthetic peptides have been proposed as analogs of Thymosin Beta 4 (TB4). These compounds were designed to be used as immunostimulants. Among them, a 16 amino acid sequence corresponding to the active site of Thymosin beta 4 has been identified to be a potent immune stimulator, when it was incorporated into protein carriers (Zwack, G. E., et al., J. Immunol. 147:3655-65, 1991).
TB4 derived peptides are also active for inducing T-cell proliferation. These peptides consist of the active domain of TB4, namely the 16 amino acid sequence including one disulphide bridge. These peptides (TB4-1 to TB4-4) showed a higher antigenicity and better immunogenicity than the native peptide and the synthetic peptides (Zwack et al., 1991). The TB4-1 to TB4-4 derived peptides have been described in European Patent application 0 686 794 and its counterpart U.S. Pat. No. 5,869,552. It is also disclosed that these peptides (TB4-1 to TB4-4) may be combined with other immunogenic compounds, in particular with monoclonal antibodies, to produce vaccine formulations.
A series of TB4 derived peptides was synthesised by several research groups. For example, Wang et al. (Wang, Q., et al., Proc. Natl. Acad. Sci. USA. 93:11963-8, 1996) showed that TB4 peptides (1 and 3) displayed enhanced antigenic properties than the native TB4 peptide, with potent activation of both CD4+ and CD8+ T-lymphocytes. Also, a study using a 15 amino acid peptide (TB4-2) showed a similar immunological activity (Kozinowski, S., et al., Faseb J. 10:933-9, 1996). Further, a series of TB4 derived peptides with two additional cysteine residues, (TB4-1 to TB4-4), (Wang, Q., et al., Immunology 90:1-10, 1996), have been reported to show higher immunogenicity than TB4 peptides (1 and 3) (Wang, Q., et al., Proc. Natl. Acad. Sci. USA 93:11963-8, 1996), however, their activity as vaccine adjuvant has not been evaluated. In this regard, Wang et al. (1996) reported that one of the analogs, TB4-3, enhanced the antigen-specific production of antibodies in a murine model.
Moreover, Thymosin Beta 4 derived peptides have also been developed and used to produce a pharmaceutical composition for use as a vaccine adjuvant. Such vaccines or adjuvants are useful in stimulating a rapid and massive generation of a wide spectrum of antibodies, and have potential application in the prevention or treatment of infectious and neoplastic diseases. The human TB4 gene contains two in frame methionine codons at nucleotide positions 14 and 397 of the mRNA sequence, resulting in the production of a 14 amino acid amino terminal fragment (amino acid positions 1-14) and a 397 amino acid peptide (positions 15-401).
These are the precursors to the disulphide-bridged 16 amino acid peptides (TB4-1 to TB4-4) which constitute the active domain of Thymosin beta 4 (TB4) protein. In an earlier study, the cloning of the human TB4 gene into pT7BlueT was described by Witt-Eggeling et al. (Witt-Eggeling, M. et al., J. Biol. Chem. 268:9771-78, 1993). According to this paper, the human TB4 gene consists of two introns and three exons. The first and second exons encode the methionine and the active domain, respectively.
The translation of the methionine codons generates an amino-terminal fragment (amino acids 1-14) with a predicted molecular weight of 14.0 kDa, whereas the translation of the active domain produces the 397 amino acid peptide (amino acids 15-401). Thus, the present invention provides for the production of an active domain from a 397 amino acid TB4 protein with a potential of improving the immune function in patients suffering from various immunosuppressive diseases. Therefore, there is a great need in the art for novel TB4 derived peptides that can enhance the immune function and stimulate a rapid and massive generation of a wide spectrum of antibodies. There is also a need for pharmaceutical compositions or adj