THYMOSIN ALPHA 11

Abstract: A polypeptide useful in the reconstitution of immune functions in thymic deprived or immunodeprived warm-­ blooded mammals of the formula AcSer-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-­ Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-­ Asn-Gly-Arg-Glu-Ala-Pro-Ala-AsnOh (I) and pharmaceutically acceptable salts thereof as well as pharmaceutical compositions containing them.

Description: Thymosin all Thymosin fraction 5, known for example from U.S. Patent 4,082,737, is a potent immunopotentiating preparation and can act to reconstitute immune functions in thymic deprived and/or immunodeprived individuals. Ongoing clinical trials with thymosin fraction 5 suggest that it is effective in increasing T cell numbers and normalizing immune function in children with thymic dependent primary immunodeficiency disease and can increase T cell numbers in immunodepressed cancer patients. The first active peptide isolated and characterized from thymosin fraction 5 has been termed thymosin 1' The isolation and characterization of this peptide is described for example in U.S. Patent 4,079.127. Synthesis of thymosin al 1 by solution and solid phase synthesis is described in U.S. Patent 4.148,788. Additionally the synthesis of thymosin al by solution phase procedures is shown in U.S. Patent 4.116,951. Thymosin al has been found to be one or more orders of magnitude more active than fraction 5 in several in vitro and in vivo assay systems designed to measure T cell differentiation and function and is currently in the clinic to determine its efficiacy in the treatment of immunodeficiency diseases, of immunodepressed cancer patients and in the prevention of opportunistic infections in immunosuppressed patients. The present invention relates to the isolation and first complete structural determination of a new polypeptide (thymosin o11) isolated from thymosin fraction 5, to pharmaceutically acceptable acid and base addition salts thereof and to pharmaceutical compositions containing this compound or its salts. Thymosin a has been found to have the same qualitative and quantitative biological activity as thymosin al in in vivo assay systems designed to measure T cell differentiation and function. Thymosin all is a polypeptide of 35 amino acid residues. the first 28 of which are identical to thymosin al. Thymosin all has the following amino acid sequence: AcSer -Asp-Ala-Ala-Va l-Asp-Thr -Ser-Ser-Glu- I le-Thr-Thr- Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu- Asn-Gly-Arg-Glu-Ala-Pro-Ala-AsnOH wherein Ac is an amino terminal acetyl group. Thymosin all was isolated from calf thymus fraction 5 by a combination of preparative isoelectric focusing (see Hannappel et al., Proc. Natl. Acad. Sci. USA 79, 1708-1711 (1982)) and HPLC (see Stein and Moschera, Methods in Enzymology 79. 7-16 to9813). Thymosin fraction 5 prepared in accordance with known procedures, see e.g. U.S. 4.079.127, was electrofocused for 17 hours at a maximum current of 20mA and a maximum voltage of 1.1 kV. The gel bed was divided into 30 segments (fractions) with a stainless steel grid and peptides from each segment were eluted with 5 ml of water. The pH of each eluate was determined with a pH meter. For analysis of the peptides in the eluates by HPLC, aliquots were lyophilized and dissolved in a small volume of buffer A (0.2M pyridine. 1.OM in formic acid). Elution was with buffer A and a linear gradient of l-propanol. In Figure 1 results are shown for the HPLC analysis of fractions 6 and 7. Peaks a, b, and c were identified as des-(25-28)-thymosin al. thymosin al, and thymosin all respectively. Some additional thymosin &alpha;1 was recovered from isoelectric focusing fractions 4 and 5 and a small quantity of thymosin all was also present in fraction 5. Separation of peptides by HPLC was performed with an Ultrasphere ODS C18 column (5 IL. 4.6 x 250 mm, Altex Scientific) with a fluorescamine detection system as described by Stein and Moschera, supra. The run shown in Figure 1 was derived from a 2 gram batch of calf thymosin fraction 5. The peptides in peak c of the HPLC purification step from isoelectric focusing fractions 6 and 7 were combined. lyophilized and purified by recbromatography on HPLC utilizing the methodology described for Figure 1. An aliquot (600 ug) was digested with 42.9 ug of TPCK-treated trypsin in 100 ul of 0.4M pyridine, pH 7.5. After 15 hours at 250C the reaction mixture was lyophilized and the tryptic peptides were separated by HPLC using a gradient of acetonitrile (0 to 30 volume percent). Fractions (0.65 ml) were collected every minute. At 6-second intervals, 5 ul samples were diverted to the fluorescamine detector. Six fragments were recovered and identified by their amino acid composition as summarized below in Table 1. Table 1. Amino acid composition of peptides isolated from tryptic and S. aureus VB protease digests of thymosin &alpha;11 Residue T1 T2 T3 T4 T5 T6 S2 S3 S7 (68)* (53)* (57)* (60)* (32)* (66)* (2.6)+ (0.8)+ (3.7)+ Asp 0.8 1.3 1.0 1.1 2.2 1.3 1.4 1.4 Thr 2.7 Ser 2.7 Glu 1.0 1.0 3.4 3.3 1.1 2.4 4.8 1.7 Gly 1.0 1.0 1.4 1.1 1.9 Ala 2.0 1.0 1.0 1.9 2.6 2.7 1.0 Val 1.9 1.5 1.1 1.0 Ile 1.0 Leu 0.8 Lys 2.1 1.0 0.2 1.0 2.9 Arg 1.0 1.1 1.0 1.0 0.5 Pro 1.3 nd** nd** nd** Calculated based on assigning a value of 1.0 for the residue as underlined. *Nanomoles recovered from a digest of 200 nanomoles of thymosin &alpha;11. +Nanomoles recovered from a digest of 8.7 nanomoles of thymosin &alpha;11. **Not determined. Peptides T6, T3 and T1 were identical to peptides deri ved from residues 1-140 15-17 and 18-20, respectively, of thymosin &alpha;1. Paptides T4 and T5 were similar in amino acid composition1 differing only in the presence of lysine in peptide T4, Their composition indicated that they corresponded to residues 20-28 of thymosin al, plus glycine and arginine. Since peptide T5 did not contain lysine, it was concluded that arginine must be located at the COOH terminus of the peptide (ses Figure 2). The tryp- tic digest contained an additional peptide fragment (T2) which was not present in tryptic digests of thymosin &alpha;1. This new peptide fragment contained no lysine or arginine, and must therefore have arisen from the COOH-terminus of thymosin all Edmann degradation of tryptic peptide T2 yielded the sequence Glu-Ala-Pro-Ala-Asn-OH. This data is summarized in Table 2. Table 2 Edman degradation of tryptic peptide T2 Nanomoles of Subtractive method, Recovered after PTH peptide recovered amino acid composition hydrolysis of the amino acid after each step of residual peptides*) anilinothiazoli- identified+) Step of degradation none(nmol)**) Glx Ala Pro Ala Asx 0 (35.3)***) 1.3 1.2 1.7 1.2 1.0 1 28.8++) 0.1 1.1 1.4 1.1 1.0 Glu (6.9) Glu 2 30.6++) 0.2 0.1 1.1 1.2 1.0 Ala (5.9) Ala 3 36.7++) 0.4 0 0.6 1.2 1.0 Pro (2.6) Pro 4 30.3++) 0.3 0 nd 0.4 1.0 Ala (2.8) Ala *) Amino acid compositions of an aliquot of thymosin &alpha;11 or of aliquots from the aqueous phase after each step of Edmann degradation. The results are presented as ratios to the quantity of aspartic acid. Half the total for alanine was arbitrarily assigned to each alanine residue in the sequence for the first 2 steps. **) An aliquot for each anilinothiazolinone was removed before cyclization and hydrolyzed for amino acid analysis. ***) This quantity was used for the degradation procedure. +) The PTH amino acids obtained at each step of the degradation were identified by HPLC. ++) Estimated from the results of amino acid analysis after acid hydrolysis. Asparagine was recovered as the free amino acid after the fourth step of the Edmann procedure Localization of peptide T2 at the COON terminus of thymosin all was confirmed by digestion of the later with carboxypeptidase (Y) which released approximately one equivalent of asparagine, followed by alanine (2 equivalents) and proline (one equivalent). The location of arginine at position 30 was confirmed by the isolation of a major fragment containing arginine after digestion of thymosin a with S. aureus V8 protease (peptide S7 in Table 1). The amino acid composition of this peptide corresponded to that predicted for residues 26-31 of thymosin all including the last four residuee of thymosin al, plus the first three amino acid residues, glycine, arginine and glutamic acid, found in the COOH-terminal extension of thymosin all. Smaller quantities of two of the fragments, whose amino acid composition corresponded to residues 19-35 (peptide S3) and 25-35 (peptide S2) of thymosin all were also isolated from the S. aureus protease digests (Table 1 and Figure 2). The results establish thymosin all as containing the thymosin al sequence plus seven additional amino acids as the COOH-terminus. The biological activity of thymosin al can be determined by utilizing in vivo assays known in the art. Thus, for example, inbred strains of mice are known to vary in their susceptability to infection with C. albicans. Thus, mice of such strains as C3 H/HeJ or CBA/CaJ are highly susceptible to infection, whereas mice of such strains as C57 Bl/lOSNJ or C57Bl/KsJ were highly resistant to challenge. Since resistance to infection with C. albicans is associated with cell-mediated processes, and therefore with T-lymphocytes, thymic hormones should have an effect on the host response. Thymosin fraction 5 and some peptides derived therefrom have been found to enhance maturation and replication of T-lymphocytes (Goldstein et al., Rec. Progress in Hormone Research 37, 369-415 (1981)) and accordingly should influence the resistance of a susceptible murine strain, such as C3H/HeJ, to infection with C. albicans. Thymosin fraction 5, thymosin CLi or all was injected daily i.p. in graded doses into three different groups of mice. beginning two days before intravenous challenge with 4 x 104 cells of Candida albicans. In comparison with control mice, all three thymic derivatives provided protection. The results are summarized in Table 3 below. Table 3. Effect of thymosin fraction 5 and thymic peptides on the growth of Candida albicans in C3H/HeJ mice Thymosin fraction 5 Thymosin &alpha;1 Thymosin &alpha;1 Dose C. albicans Dose C. albicans Dose C. albicans ng/mouse cell count* ng/mouse cell count* ng/mouse cell count* 2560 8500 80 5870 80 4200 5120 440 160 190 160 510 10240 320 320 780 320 320 20480 1600 640 1410 640 1260 Mice were treated daily with the indicated doses of thymosin fraction 5, thymosin &alpha;1 or thymosin &alpha;11 and challenged with 4 x 104 cells of C. albicans two days after the start of treatment *Three mice from each set were sacrificed on days 7, 14 and 21 after infection and the values represent the average number of organisms in the left kidneys of the nine mice in each set. The polypeptides, thymosin &alpha;1 and thymosin &alpha;11, were approximately equal in potency, be inc most active in daily doses of 160-320 ng per mouse i.p. Since the optimum dose for fraction 5 was 5-10 Vg, the peptides were about 30 times more portent than fraction 5 in their ability to induce resistance to infection with C. albicans. Thus, thymosin &alpha;11, in analogy to thymosin al can be used as therapeutical agent a especially as an agent useful in the reconstitution Of immune functions in thymic deprived or immunodeprived warm-blooded mammals. It may be administered in the form of pharmaceutical compositions. i.e. in mixture with a suitable pharmaceutically acceptable carrier and, if desired, further adjutants to warm-blooded mammals by parenteral application either intravenously. subcutaneously or intramuscularly. The compound is a potent immunopotentiating agent with a daily dosage in the range of about 1 to 100 g/kg of body weight for intravenous administration. Obviously the required dosage will vary with the particular condition being treated, the severity of the condition and duration of the treatment. A suitable dosage form for pharmaceutical use is 1 mg of lyophylized thymosin Qll per vial to be reconstituted prior to use by the addition of sterile water or saline. Also included within the scope of the present invention are the pharmaceutically acceptable salts of thymosin &alpha;11 such as the sodium or potassium salts or salts with strong organic bases such as guanidine. In addition, the counter ions of these cations as well as of lysine residues such as the chloride, bromide. sulphate, phosphate, maleate, acetate, citrate, benzoate, succininate, ascorbate and the like, may be included in the preparation. It is also within the scope of the present invention to modify the sequence of thymosin all by single amino acid changes or by derivatizing the carboxy terminus by ester or amide formation. Also within the scope of the invention is desacetyl thymosin all which can be produced microbially, using recombinant DNA techniques, e.g. a synthetic gene which is introduced into a suitable microbial expression vehicle. After transformation with such an expression vehicle microorganisms or mammalian cells will be able to express desacetyl thymosin all under suitable conditions. Desacetylthymosin 11 would have the same biological activity as thymosin all by analogy to the relationship between thymosin CLi and desacetylthymosin al.

Claims: aLArMS: 1. Thymoin all, a polypeptide of the sequence AcSer-Asp-Ala-Ala-Val-Asp-=Thr-Ser-Ser-Glu-Ile-Thr-Thr Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala Glu-Asn-Gly-Arg-Glu-Ala-Pro-Ala-AsnOH (I) and pharmaceutically acceptable acid and base addition salts thereof. 2. The peptide of formula I as claimed in claim 1 essentially free of other thymic polypeptides. 3. A compound as claimed in claim 1 or claim 2 for use as therapeutical agent. 4. A compound as claimed in claim 1 or claim 2 for use as an agent reconstituting immune functions in thymic deprived or immunodeprived warm-blooded mammals. 5. Pharmaceutical compositions containing a compound as claimed in claim 1 or claim 2 and a equitable, pharmaceutically acceptable carrier. 6. Compositions useful in the reconstitution of immune functions in thymic deprived or immunodeprived warm-blooded mammals containing a compound as claimed in claim 1 or claim 2 and a suitable, pharmaceutically acceptable carrier.

Inventors: HORECKER, BERNARD LEONARD
Applicants: F. HOFFMANN-LA ROCHE & CO. AKTIENGESELLSCHAFT
Application year: 1984
Main classification: C07K7
IPC codes: A61K38, C07K14