Short Communication Mediators of Inflammation, 10, 89–92 (2001)

Two [Met(0)6]deacetyl-thymosin beta4 analogs containing Phe(4F) or Tyr(Me) at position 12 were synthesized by the manual solid-phase method, and their anti-inflammatory effect on carrageenin-induced edema in the mouse paw was studied. Fluorination of the para-position of Phe12 resulted in a marked antiinflammatory effect on carrageenin-induced edema in the mouse paw compared with that of our synthetic [Met(0)6]deacetyl-thymosin beta4, but the other analog, [Met(0)6, Tyr(Me)12]deacetyl-thymosin beta4, showed a marked reduction of the anti-inflammatory effect.


Introduction
Thymosin b 4 consists of 43 amino acid residues ( Fig.  1) with a molecular weight of 4963 and an isoelectric point of 5.1. 1 The N-terminus of the peptide is blocked by an acetyl group. This peptide exhibits important activities in the regulation and differentiation of thymus-dependent lymphocytes.
Recently, Young et al. 2 reported that [Met(0) 6 ]thymosin b 4 (Met(0), methionine sulfoxide) is generated by monocytes in the presence of glucocorticoids and acts as a signal to inhibit an inflammatory response, and [Met(0) 6 ]thymosin b 4 was a potent inhibitor of carrageenin-induced edema in the mouse paw. [Met-(0) 6 ]thymosin b 4 may have value in anti-inflammatory drug therapy, with great potent advantages over existing nonsteroidal drugs that alleviate the distressing symptoms of inflammation without preventing the tissue damage. Therefore, [Met(0) 6 ]thymosin b 4 might be able to reproduce the considerable benefits of glucocorticoids originally seen in rheumatoid arthritis, but without the subsequent disabling steroid toxicity.

Solid-phase peptide synthesis
Peptide synthesis was performed manually by the stepwise solid-phase method with a hand-made peptide synthesizer, using the base-labile Fmoc group for protecting the b -amino groups, and such acid-labile groups as tert-butyl for the hydroxy and carboxy groups, tert-butyloxycarbonyl for the b -amino groups of Lys, and sulfoxide for Met.
After the deprotection, the resin was removed by filtration and the filtrate was evaporated under reduced pressure, and the residue was solidified by addition of anhydrous ether to give a crude peptide. The resulting powder was dissolved in H 2 O (6 ml). The solution was treated with Amberlite CG-4B (acetate form, approximately 3 g) for 30 min, and filtered by suction and evaporated in vacuo. The residue was dissolved in a small amount of 1% acetic acid and then applied to a column of Sephadex G-25 (2.3 ´96 cm), which was eluted with the same solvent. Individual fractions (5 ml each) were collected and absorbancy at 260 nm was determined for each fraction. The fractions corresponding to the front main peak were combined and the solvent was removed by lyophilization. The peptide was further purified by semi-preparative PR-HPLC. The semipreparative PR-HPLC was performed on a Nucleosil C18 column (250 ´10 mm I.D.; 7 m m particle size; Marcherey Nagel). Solvent A was 0.05% TFA in water and solvent B was 60% acetonitrile in solvent A. A linear gradient was applied from 10 to 50% B during 50 min, at a flow rate of 3.0 ml/min. Detection of the peptide was set at 230 nm. The major peak was lyophilized to give the purified product. Overall yields of the two peptides were 5.9% ([Met(0) 6 , Phe-(4F) 12 ]deacetyl-thymosin b 4 ) and 6.1% ([Met(0) 6 , Tyr(Me) 12 ]deacetyl-thymosin b 4 ), respectively, based on the C-terminal Ser loaded on the resin. Homogeneity of the peptides was checked by TLC, analytical HPLC, FAB-MS, and amino acid analysis after 6 N HCl hydrolysis, The physicochemical data of the synthetic analogs are presented in Tables 1 and 2.

Bioassay
Carrageenin-induced inflammation was initiated in BALB/C mice as described elsewhere. 5   (obtained from protected deacetyl-thymosin b 4 3 without reduction treatment), and [Met(0) 6 , Phe-(4F) 12 ]deacetyl-thymosin b 4 and [Met(0) 6 , Tyr(Me) 12 ]deacetyl-thymosin b 4 , was administered 30 min before (intraperitoneal injection), coincident with (intra-paw injection) and 6 h after (intraperitoneal injection) carrageenin injection into the right hind footpad. The change in footpad thickness between right and left hindlimbs was assessed using dial calipers by two observers 'blinded' to the treatment status of the mice. Control carrageenin-injected mice received phosphate-buffered saline at similar times. We judged our synthetic peptide had a positive effect when we found more than 80% of footpad swelling was suppressed (Tables 3 and 4).

Results and discussion
In our preceding paper, 3 we reported that Phe 12 residue of deacetyl-thymosin b 4 is one of the struc-tural essentials for immunological activity on the impaired blastogenic response of uremic T lymphocytes. One of our synthetic analogs, 3,4 [Phe(4F) 12 ]deacetyl-thymosin b 4 , exhibited stronger immunological activity than that of our synthetic deacetyl-thymosin   carrageenin-induced edema in the mouse paw is presented in Table 3.
On the contrary, another analog, [Met(0) 6 , Tyr (Me) 12 ]deacetyl-thymosin b 4 , which has an electrondonating group -OCH 3 on the para-position of the aromatic ring, showed a much weaker anti-inflammatory effect than that of our synthetic [Met(0) 6 ]deacetyl-thymosin b 4 . Our synthetic [Met(0) 6 ]deacetylthymosin b 4 at a concentration of 20 m g induced suppression equivalent to the suppression induced by 0.5 mg/kg dexamethasone, a potent anti-inflammatory steroid, and one of our two analogs, [Met(0) 6 , Phe(4F) 12 ]deacetyl-thymosin b 4 , showed the same suppression activity at the concentration of 5 m g induced suppression equivalent to the suppression induced by 0.5 mg/kg dexamethazone, which means that suppression activity of this analog is about fourfold stronger than that of [Met(0) 6 ]deacethylthymosine b 4 (data not shown). These results seem to suggest that aromaticity at the 12 position of [Met-(0) 6 ]deacetyl-thymosin b 4 plays significant roles for anti-inflammatory activity on carrageenin-induced edema in the mouse paw, and modification of the Phe residue of thymosin b 4 could produce more potent analogs capable of anti-inflammatory effects on inflammatory diseases.