Drug – bio-affecting and body treating compositions – Preparations characterized by special physical form – Capsules
Reexamination Certificate
2001-01-23
2003-08-05
Page, Thurman K. (Department: 1615)
Drug, bio-affecting and body treating compositions
Preparations characterized by special physical form
Capsules
C424S464000, C424S489000, C424S422000, C424S434000, C424S435000, C424S436000, C514S002600, C514S012200
Reexamination Certificate
active
06602519
ABSTRACT:
The present invention relates to a peptide factor isolated from steroid-treated monocytes. More particularly the invention relates to a peptide factor which can be used to replace steroid therapy.
Steroids are effectively used for anti-inflammatory diseases, such as asthma, eczema, allergic reactions, and rheumatic diseases such as rheumatoid arthritis. However, steroids have serious side effects and are therefore only used in cases where non-steroidal anti-inflammatory drugs are not effective.
Monocytes are important immune effector cells that play a fundamental role in cellular immunity. In addition to their antigen-presenting and phagocytic activities at the sites of inflammation, peripheral blood mononuclear cells are also involved in the synthesis and release of a variety of pro-inflammatory enzymes and polypeptide cytokines which modulate neutrophil responses. The production of these components can be suppressed by glucocorticoids and this has been suggested as the basis for their anti-inflammatory action.
The effect of steroid-induced factors on neutrophil migration is primarily of interest in elucidating anti-inflammatory mechanisms. Corticosteroids down regulate the synthesis of many pro-inflammatory mediators (Lew et al 1988; Almawi et al 1991; Standford et al 1992) but some of their actions can be interpreted in terms of up-regulation of anti-inflammatory mediators.
The neutrophil migration stimulating activity of steroid induced factors suggests that dispersive locomotion tends to prevent cells collecting at a focus and this may be important in terminating inflammatory responses.
Stevenson (1973, 1974, 1978) demonstrated that human monocytes when incubated in the presence of antiinflammatory corticosteroids released a protease sensitive factor that enhanced the migration of neutrophils from a cell pellet contained in a short capillary tube.
Later studies demonstrated that the phenomenon of stimulated neutrophil migration was also observed with leucocytes from patients receiving steroid therapy.
Recently, Chettibi et al (1993, 1994) have investigated the steroid induced stimulatory effect on neutrophil migration using an automated cell tracking assay enabling study of the behaviour of cells migrating on protein-coated glass coverslip.
These Studies Determined:
1. Steroid-treated monocyte supernatant (STMS) causes a dramatic increase in the speed of locomotion of human neutrophils and a significant decrease in their adhesion to protein-coated glass. In contrast, control monocyte supernatants have a smaller effect on the speed of locomotion, but cause a large increase in adhesiveness.
2. The supernatant activity was produced equally well in the presence or absence of serum after 24 h culture at 37° C. with 10
−6
M dexamethasone.
3. The effect of the steroid-treated monocyte supernatant on the speed of locomotion of human peripheral blood neutrophils was not altered by rabbit polyclonal antisera against lipocortins 1-6.
4. Rabbit anti-interleukin-8 antibody which blocked the effect of IL-8 on the speed of locomotion of neutrophils did not antagonize the locomotion stimulating action of steroid-treated monocyte supernatant.
5. The exocellular release of this factor(s) by human mononuclear leucocytes suggests that it may be an in vivo mediator of the anti-inflammatory effect of glucocorticoids.
However, there is no disclosure of what the active agent(s) in STMS might be.
Huff T. et al. (1995) and Heintz D. et al. (1994) describe studies involving beta-thymosins and how they interact with G-actin in a biomolecular complex and inhibit the polymerisation to F-actin under high salt conditions. The oxidised form of thymosin &bgr;4 is disclosed as inhibiting actin polymerisation, however, only at a 20-fold higher concentration than thymosin &bgr;4. Neither document however implicates any medical role for oxidised thymosin &bgr;4. In fact the papers appear to teach away from a positive role for oxidised thymosin &bgr;4.
U.S. Pat. No. 5,578,570 (Goldstein et al.) discloses a method of treating septic shock by administering thymosin &bgr;4. There is no disclosure however of oxidised thymosin &bgr;4 or suggestion that this may have a role in treating septic shock.
It is an object of the present invention to provide a replacement to steroid therapy.
The present invention is based in part on the observations by the present inventors that the factor associated with neutrophil locomotion is an oxidised form goof thymosin &bgr;4.
According to a first aspect the present invention provides use of oxidised thymosin &bgr;4 or physiologically active variant thereof in therapy.
Typically oxidised thymosin &bgr;4 is a form of thymosin &bgr;4 in which a methionine residue, 6 amino acids from the N-terminus, (Met6), is oxidised such that the residue is converted to methionine sulphoxide. Moreover, the methionine residue (Met6) may be further oxidised to the methionine sulphone and this as such is also encompassed by the present invention. Other modifications of the methionine residue may also be evisaged, such as complexing the sulphur with metals, which may result in an active form of thymosin &bgr;4 similar to the oxidised form described herein.
It is understood that the oxidised thymosin &bgr;4 may be obtained for example by reacting native thymosin &bgr;4 under oxidising conditions, for example by treating with hydrogen peroxide, to form oxidised thymosin &bgr;4. Thus native thymosin &bgr;4 may first be obtained and thereafter oxidised to the oxidised form.
It has been observed that samples of native thymosin &bgr;4 may contain low levels, such as 10%, of oxidised thymosin &bgr;4 thought to be as a result of auto-oxidation. The present inventors however are the first to associate the oxidised form of thymosin &bgr;4 with a physiological activity. Generally speaking therefore the present invention provides the use of purified oxidised thymosin &bgr;4. Typically the present invention provides use of preparations of purified oxidised thymosin &bgr;4 which comprise at least 30%, preferably 60%, more preferably 80%, most preferably 90%, oxidised thymosin &bgr;4 with the residual portion accounting for non-oxidised thymosin &bgr;4. Preferably however the preparations of oxidised thymosin &bgr;4 comprise substantially all oxidised thymosin &bgr;4 (ie. substantially no non-oxidised thymosin &bgr;4).
Thymosin &bgr;4 in an oxidised or non-oxidised form may be obtained from any suitable source, for example from steroid treated monocytes. Moreover, the thymosin &bgr;4 may be derived from any suitable species, but is typically of mammalian origin, such as bovine, equine, murine or human origin. It is to be noted that bovine, equine, murine, rat and human thymosin &bgr;4 are all identical in sequence. Thus, for example, bovine thymosin &bgr;4 may provide a suitable source of thymosin &bgr;4 for subsequent oxidation and administration to other species, such as humans.
It is understood that physiologically active variants of the oxidised thymosin &bgr;4 are variants which display the same or similar physiological properties as the oxidised thymosin &bgr;4. It is to be preferred that such variants would include the oxidised methionine, but may be truncated, deleted or mutated forms thereof.
It will be understood that for the particular oxidised thymosin &bgr;4 embraced herein, variations (natural or otherwise) can exist. These variations may be demonstrated by (an) amino acid difference(s) in the overall sequence or by deletions, substitutions, insertions, inversions or additions of (an) amino acid(s) in said sequence. All such derivatives are included within the scope of this invention provided that the derivatives are physiologically active (ie. display oxidised thymosin &bgr;4 activity as defined herein). For example, for the purpose of the present invention conservative replacements may be made between amino acids, within the following groups:
(I) alanine, serine and threonine;
(II) glutamic acid and aspartic acid;
(III) arginine and lysine;
(IV) asparagine and glutamine;
(V) isoleucine, leucine
Lawrence Anthony John
Pappin Darryl John Cecil
Stevenson Robert Duncan
Young John
Di Nola-Baron Liliana
Myers Bigel & Sibley Sajovec, PA
The University Court of the University of Glasgow
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