WO1992013546A1 - Mb-40 peptide that enhances the production of interleukin-1 and modulates age-associated immune response - Google Patents

Abstract

A peptide of the formula: Met-Gln-Ile-Phe-Val-Lys-Thr-Leu-Thr-Gly-Lys-Thr-Ile-Glu-Leu-Glu-Val-Glu-Pro-Ser-Asp- Thr-Ile-Glu-Asn-Val-Lys-Ala-Lys-Ile-Gln-Asp-Lys-Ser-Gly-Ile-Pro-Pro-Asp-Gln has been isolated from thymosin fraction 5(TF-5) and synthetically produced. This peptide, which has 95 % homology with the first 40 amino acids of ubiquitin (38/40 amino acid identity) is able to stimulate and enhance the production of IL-1α by peripheral blood monocytes. The peptide can be used alone or in combination with other IL-1 or IL-2 stimulants, such as thymosin α1 to increase the production of IL-2.

Description

MB-40 A PEPTIDE THAT ENHANCES THE PRODUCTION OF INTERLEϋKIN-1 AND MODULATES AGE-ASSOCIATED

IMMUNE RESPONSE

BACKGROUND OF THE INVENTION Field Of The Invention:

This invention relates to a novel peptide and to the use thereof for stimulating interleu in-1 production and modulating age-related changes in lymphocytes that affect the immune response. Description Of The Prior Art:

Thymosin factor 5 (TF5) is a partially purified thymus gland preparation consisting of a large family of biologically active polypeptides with hormone-like activities. TF5 is known to modulate the immune response of immunodeficient individuals. It has recently been shown that TF5 acts on macrophages and induces the production of interleukin-1 (IL-1) in vitro and the expression of a membrane-associated form of IL-l, mIL-1, in vivo. See Hu, et al., Lymphokine Research, Vol. 8, No. 3 pp. 203-213 (1989).

Interleukin-1, a monokine, is secreted by macrophages during the process of antigen processing. IL-1 aids in the activation of T-lymphocytes, making the lymphocytes responsive to IL-2, a T-cell growth factor necessary for the production of the natural killer cells involved in immuno regulation. Recently, a membrane associated form of IL-1 (mIL-1) was isolated. Kurt Jones, et al., PNAS Vol. 82, pp. 1204 (1985); Kurt Jones, et al., Immunology, Vol. 137, pp. 10 (1986). This mIL-1 membrane associated form of IL-1 is biologically active and probably plays a significant role in immuno stimulation of T-lymphocytes in local tissues such as the lymph nodes, joints and skin. The expression of mIL-1 appears to be quantitatively linked to activation of T-lymphocytes. It has also been reported that IL-1 stimulates the production of IL-6, a cytokine having multiple roles in inflammatory immune responses, including enhancements of T-cell activation and stimulation of B-cell differentiation. Kishimoto, T. 1989n, The Biology of Interleukin-6. Blood 74:1. In addition, IL-6 stimulates the release of anterior pituitary hormones. TF5 has been shown to increase the production of IL- 2, Zatz, et al., PNAS(1984) and the expression of IL-2 receptors in normal human peripheral blood lymphocytes, Serrate, et al.. Immunology (1987). The effects of TF5 are eliminated, however, if macrophages are removed from the lymphocyte preparation, suggesting that TF5 may act directly on the macrophages by inducing the production of IL-1 which in turn activates T-cells to secrete IL-2.

Studies have shown that lymphokines, such as IL-1 and IL-2, play a central role in the development, maintenance, and regulation of immune responses. There is also evidence that there is a deficiency of macrophages and IL-1 production in aging. Zatz and Goldstein, Gerontology, 31:263-277 (1985) . It has further been demonstrated that there is a link between the endocrine thymus and the hormones of the adrenal glands. TF5 has been found to stimulate the production of ACTH, cortisone and b-endorphin in primates indicating that thymosin factors may be critical in regulating factors of brain hormonal activity and thus may play a major role in senescence of immunity. Healy, et al.. Science 222:1355 (1983). It has become increasingly evident that IL-1 and IL-2 are critical in modulating lymphoid cell interactions and that aberrations of IL-1 or IL-2 production or activity may play an important role in the senescence of the immune response.

Various methods are known to stimulate the immune system both in vivo and in vitro. Administration of certain thymus hormones such as thymopoietin (U.S. Patent No. 4,002,740 and U.S. Patent No. 4,077,949), induces the differentiation of T-cells. Due to the restricted natural resources of such hormones and the characteristic instability of thymopoietin in purified form extensive studies were initiated to determine the active site of thympoietin. Small peptides and analogs of thymopoietin have been synthesized that possess all of the biological properties of the complete hormone, U.S. Patent No. 4,190,646. Other methods of stimulating the immune system have included the administration of serum-free, mitogen free IL-1 and serum free, mitogen-free IL-2, U.S. Patent No. 4,681,844. This method is cumbersome in that it involves the culturing and manipulation of peripheral blood mononuclear cells and has the disadvantage that purified IL-1 and IL-2 have a short half- life.

IL-1 and IL-2 are essential for proper immune system function. Abnormalities in thymus function result in alterations of immune reactions which are the direct result of thymic hormone deregulation.

SUMMARY OF THE INVENTION The present inventor has isolated, purified and characterized a biologically active polypeptide from TF5 that has the ability to induce the expression of a membrane- associated interleukin-1(mIL-1) and modulate the effects of age on graft versus host disease (GVHD) . This peptide, hereinafter MB-40, is a 40 amino acid peptide with molecular weight of 4,443 Daltons and an isoelectric point of 5.2 as determined by isoelectric focusing at a pH range of 3.5-9.2. The amino acid sequence of MB-40 is:

Met-Gln-Ile-Phe-Val-Lys-Thr-Leu-Thr-Gly-

Lys-Thr-Ile-(Glu)-Leu-Glu-Val-Glu-Pro-Ser-

Asp-Thr-Ile-Glu-Asn-Val-Lys-Ala-Lys-Ile- Gln-Asp-Lys-(Ser)-Gly-Ile-Pro-Pro-Asp-Gln.

Peptide MB-40 and human thymosin βl (ubiquitin) share amino acid sequence homology in residues 1-40 of ubiquitin, with only two amino acid differences at positions 14 and 34 of ubiquitin. Ubiquitin, a 76 amino acid protein- is one of the most highly conserved proteins among eucaryotes; only three amino acid substitutions have occurred between yeast and human ubiquitins. It is not known whether MB-40 is a breakdown product of free ubiquitin or chromosomal protein A24 (a histone H 2A-ubiquitin complex) or whether it is a distinct protein. MB-40 can be used to induce the production of interleukin-1 by human peripheral blood lymphocytes in vitro by incubating peripheral blood monocytes with an appropriate concentration of MB-40. Synthetically produced MB-40 significantly increases the level of IL-lα production beyond that observed with the partially purified TF5. Intraperitoneal injection of TF5 induces the expression of a membrane- associated IL-l(mΙL-l) on mouse peritoneal cells and presumably this is also true for the synthetic peptide MB-40. Thus, the invention encompasses the use of the peptide, MB-40, alone and in combination with other IL-1 inducing agents or thymic peptides in the therapeutic treatment of humans and animals. In a preferred embodiment of the invention, a therapeutically effective concentration of MB-40 can be administered to a human or an animal in order to stimulate the in vivo production of IL-lα. MB-40 may be used alone or in concert with other thymic peptides or agents that stimulate the production of IL-lα and ultimately increase the level of IL-2. The present invention provides a novel peptide having the ability to induce endogenous IL-lα production. The peptides of this invention have from 20 to 60 amino acids including the whole or part of the peptide having the amino acid sequence represented by the formula I:

Met-Gln-Ile-Phe-Val-Lys-Thr-Leu-Thr-Gly- Lys-Thr-Ile-(Glu)-Leu-Glu-Val-Glu-Pro-Ser- Asp-Thr-Ile-Glu-Asn-Val-Lys-Ala-Lys-Ile- Gln-Asp-Lys-(Ser)-Gly-Ile-Pro-Pro-Asp-Gln (I) or an analog thereof having part or all of the primary sequence and biological activity of the peptide of the given sequence. The invention also includes peptides having the whole or part of the peptide having the amino acid sequence represented by the formula I covalently linked at its carboxyl terminus to the NH₂ terminus of the peptide having the amino acid sequence represented by formula II.

Gln-Arg-Leu-Ile-Phe-Ala-Gly-Lys-Gln-Leu- Glu-Asp-Gly-Arg-Thr-Leu-Ser-Asp-Tyr-Asn- lie (II) or an analog thereof having part or all of the primary sequence of the peptide of the given sequence. The invention also provides a method for modulating the affects of age on graft versus host disease by administering an effective amount of peptide having the sequence of formula I or a fragment or analog thereof to an animal in need of such treatment.

Related to this method is the treatment of individuals having dysfunctional immune systems due to the effects of aging or loss of thymic hormone production by administering to such individuals an effective amount of the peptides either alone or in combination with other immuno stimulatory agents.

The invention also provides method for inhibiting the release of IL-6 both in vitro and in vivo. Exposure of cultured cells capable of IL-6 production to an effective amount of peptide having the sequence of Formula I, II or a fragment or analog thereof inhibits the release of IL-6 by said cells in vitro. A method for modulating the release of IL-6 in vivo is provided by administering an effective amount of peptide having the sequence of formula I, formula II or a fragment or analog thereof to an animal in need of such treatment.

The present invention also provides pharmaceutical compositions containing the biologically active peptide, with or without other immuno stimulating agents in combination with a major amount of pharmacologically acceptable liquid or solid carrier according to conventional pharmaceutical techniques. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a diagrammatic representation of the relationship between the invention peptide MB 40, ubiquitin and histone H 2A.

Figure 2 is a chro atograph of the reverse-phase high performance liquid chromatographic (RP-HPLC) separation of thymosin fraction 5(TF5) on a 300 x 50 mm Delta-pak C₁₈ column. Figure 3 is a chromatograph obtained from the rechromatography of fraction 50 (F-50) from pool MB 7 of the RP-HPLC chromatograph of Figure 2 on a 150 x 39 m Delta-pak C₁₈, 300 A, 5μ column. Figure 4 is a chromatograph of fraction 60 (F60) from the RP-HPLC chromatograph of F50 in Figure 3.

Figure 5 is a photograph of a Coomassie Blue stained isoelectric focusing gel, pH 3.5-9.5, Lane 1: LKB/Pharmacia IEF pH markers; Lane: 2: 20 μg MB-40 Lane 3: 75 μg thymosin fraction 5 and Lane 4: 10 μg ubiquitin.

Figure 6 is a photograph of a Coomassie Blue stained SDS-polyacrylamide gel of peptide MB-40; Lane 1: low molecular weight markers from Bio-Rad; Lane 2: 2 μg peptide MB-40. Figure 7 is a bar graph showing the effects of various concentrations of TF5, ubiquitin and HPLC-purified MB- 40 on the induction of IL-1 in PBM'S.

Figure 8 is a chromatograph of the crude synthetic peptide MB-40 produced as shown in Example 3 from RP-HPLC separation on a 150 x 3.9 mm -I.D. Delta-pak C₁₈ column.

Figure 9 is a chromatograph obtained from the rechromatography of purified synthetic peptide MB-40 (fraction 29 from the RP-HPLC chromatograph of Figure 8) .

Figure 10 is a photograph of a Coomassie Blue stained IEF gel. Lane 1: LKB/Pharmacia IEF pH markers, pH 3-10 Lane 2: 20 μg synthetic MB-40 and Lane 3: 20 μg RP-HPLC-purified MB-40.

Figure 11 is a Coomassie Blue stained 10% SDS- polyacrylamide gel. Lane 1: molecular weight markers Lane 2: 20 μg synthetic MB-40 and Lane 3: 20 μg RP-HPLC purified MB- 40.

Figure 12 is a bar graph showing the effects of various concentrations of MB-40 and TF5 in the in vitro assay for IL-1 production. Figure 13 is bar graph showing the effects of synthetic peptide MB-40 and TF5 on GVHD response by measuring the lysis of EL 4 target cells (150:1 Panel A) or immunoglobulin production (Panel B) relative to normal control. Figure 14 is a bar graph showing the effects of synthetic peptide MB-40 on the release of IL-6 from medial basal hypothalami (MBH) in vitro. DETAILED DESCRIPTION OF THE INVENTION The present invention is concerned with a new peptide having therapeutic value in various areas, therapeutic compositions containing this peptide and methods for use thereof. The present invention is the result of research on hormones of the thymic gland and their effects on other endocrine tissues and the immune system. Accumulated information has led to the conclusion that macrophages play an important role in all immune responses and that hormonal modulation of macrophage activity is essential for proper immune responses. It appears that thymic hormones and other thymic factors may modulate macrophage activity and thus indirectly regulate both T-cell differentiation and "thymic education." It has been suggested that the three major cellular components of the thymus, i.e., thymocytes, epithelial cells and macrophages, interact with each other such that the epithelial cells release the thymic hormones, thymosin B-4 and thymosin _lf which then act on thymocytes to induce their differentiation. (Low & Goldstein, J. Biol. Chem. , 254. 987 (1975)).

The molecular basis for interactions between the epithelial cells and macrophages has not been defined but it is suspected that the epithelial cells of the thymus secrete soluble factors capable of modulating macrophage activity. Examination of the effects of a partially purified thymic extract of bovine origin, thymosin fraction 5 (TF-5) , upon IL-lα release in vitro indicates that TF5 specifically induces human peripheral blood monocytes to secrete a factor with IL-lα activity. Antibodies to IL-lα neutralize the IT--1 activity in a dose-dependent manner indicating that TF5 specifically induces the release of IL-lα.

The specific peptide in TF5 responsible for inducing IL-1 (MB-40) has been identified, isolated and synthetically produced. MB-40 was first isolated from TF5 by reversed-phase high performance liquid chromatography (HPLC) . Details of the procedure are described in Example 1. MB-40 has also been produced using synthetic solid phase peptide synthesis, as shown in Example 3. The synthetic peptide was purified by HPLC (see Fig. 2) . The identity of the purified synthetic peptide was confirmed by amino acid composition analysis and isoelectric focusing polyacrylamide gel electrophoresis. The synthetic peptide has the same biological activity as the factor derived from TF5.

Computer analysis of the primary sequence of MB-40 using the Protein Identification Resource (PIR) computer system has established that MB-40 has homology to residues 1-40 of thymosin β_]^ or ubiquitin isolated from bovine thymus. The sequences of human and bovine ubiquitin are identical. Peptide MB-40 differs from ubiquitin only in residues 14 and 34. Ubiquitin is 76 amino acids long and is one of the most highly conserved proteins among both lower and higher eucaryotes; only 3 amino acid substitutions in the entire molecule have occurred between yeast and human ubiquitins. Ubiquitin is a component of the nuclear protein A24 complex that has been found in several mammalian tissues, including calf thymus. Protein A24 is a dimer of histone H2A and ubiquitin. In bovine thymus A24, the two protein chains are linked by an isopeptide bond between H2A Lys-119 and the Gly at the carboxyl terminus of ubiquitin. Bovine and human ubiquitin have the following amino acid sequence: Met-Gln-Ile-Phe-Val-Lys-Thr-Leu-Thr-Gly-

Lys-Thr-Ile-Thr-Leu-Glu-Val-Glu-Pro-Ser- Asp-Thr-Ile-Glu-Asn-Val-Lys-Ala-Lys-Ile- Gln-Asp-Lys-Glu-Gly-Ile-Pro-Pro-Asp-Gln- Gln-Arg-Leu-Ile-Phe-Ala-Gly-Lys-Gln-Leu- Glu-Asp-Gly-Arg-Thr-Leu-Ser-Asp-Tyr-Asn-

Ile-Gln-Lys-Glu-Ser-Thr-Leu-His-Leu-Val- Leu-Arg-Leu-Arg

Fig. 1 is a diagrammatic representation of the structure of A24 showing the relationship between ubiquitin and histone 2A and the location of the MB-40 sequence within the ubiquitin molecule. The relationship between MB-40 and ubiquitin is not understood at this time. It has been inferred from its high degree of evolutionary conservation and wide spread occurrence that ubiquitin must play a role in one or more critical functions within the cell. Ubiquitin is found in all eucaryotes, either free or attached to one of a variety of nuclear or membrane proteins. It has been ascribed to have various activities, depending on the protein to which it is covalently bound. For example, when added to T- or B-precursor cells ubiquitin induces their differentiation into mature T or B cells (Audhya, et al., 1985, Methods Enzymol. 116). At this time it is not known whether MB-40 is a degradation product of free ubiquitin, chromosomal protein A24 or possibly a unique protein. The molecular mechanisms by which MB-40 causes the induction of IL-lα release or modulates the effects of age on GVHD response are not understood at this time. However, it is anticipated that modifications of MB-40, such as amino acid additions, deletions, substitutions or combinations thereof, can be made without the loss of the peptide's desired biological activities. Therefore, any such modifications are considered to be within the scope of this invention.

For instance, the specific additions of amino acid sequences derived from ubiquitin residues 40-60 can be made sequentially to the N-terminus of MB-40. When such amino acid additions are made, they will preferably total less than 10 amino acids, as the synthesis of each amino acid becomes more difficult as the total number of amino acids approaches 50. Most preferably, no amino acids will be added to either the COOH or NH₂ terminus. Additions of l, 2 or 3 amino acids or substitution of one or both terminal amino acid residues in order to facilitate the synthesis of the peptide, e.g., by providing linkage groups or for introducing modifying groups, or for introducing tagging groups, e.g., radioactively labelled amino acid for radioimmunoassays, etc., can be advantageous as long as the modification does not result in the loss of the desired biological activity. Additions of arbitrary amino acids can be made to either the COOH or NH₂ terminus, again with preferably less than 10 residues for any given modification. Also, additions of amino acids within the MB-40 sequence may also be permissible. Amino acids may be added within the MB-40 sequence either consecutively or non- consecutively. Again, any additions must not eliminate the desired biological activity of the peptide.

Similarly, amino acids may be deleted from MB-40, as long as such deletions do not eliminate the biological activity. Deletions may actually facilitate synthesis of the peptide, by eliminating unnecessary chemical reactions in the synthesis of the peptide. Suitable additions, deletions or substitutions, that do not interfere with or alter the peptide's active site can be ascertained by the user by assaying for IL-1 stimulation of peripheral blood monocytes as described below.

Conservative substitutions of amino acids that maintain the biological activity of the peptide may also be made. Examples of substitutions, but not limited to such, include substitutions among non-polar aliphatic non-charged amino acids, including glycine, proline, valine, isoleucine, leucine and alanine. Preferred substitutions within this group include substitutions between alanine and glycine and between valine, leucine and isoleucine. Substitutions among the polar aliphatic neutral amino acids, will include serine (Ser) , threonine (Thr) , methionine (Met) , cysteine (Cys) , asparginine (Asn) and glutamine (Gin) . Substitutions may be made among the charged acidic amino acids, including aspartic acid (Asp) and glutamic acid (Glu) or among the charged basic amino acids, including lysine (Lys) and arginine (Arg) . Substitutions among the aromatic amino acids may include phenylalanine (Phe) , tryptophan (Trp) , tyrosine (Tyr) and histidine (His) .

Substitutions of amino acids from among the aforementioned groups may also be made, for example: Asp, Glu and His; Asn, Gin, Lys and Arg; Asp, Glu, Lys and Arg; Pro and

Arg; Ser, Thr, Ala, Pro and Val; and Gly, Ala, Pro, Asp and Glu. Ser and Thr is also an interchangeable group of amino acids that usually retains biological activity of the peptide. It is also considered that the scope of the present invention is inclusive of the unsubstituted peptides as well as those that are terminally substituted by one or more functional groups that do not substantially affect the biological activity of the peptide as disclosed herein. These functional groups include, but are not limited to, such substitution as acylation on the free amino group and amidation on the free carboxylic acid group.

The peptides of this invention can be prepared by conventional processes of peptide synthesis. Typical methods include an azide process, a chloride process, an acid anhydride process, a mixed anhydride process, a DCC process, an active ester process (p-nitrophenyl ester process, an N-hydroxy succinimide ester process, a cyano methyl ester process, etc.) , a carbodiimidazole process, an oxidation reduction process, a method using Woodward reagent K, a DCC/additive (HONB, HOBt, HOSu) process, etc. The preferred method of syntheses is a solid phase process.

Once the peptide has been synthesized, for example, by a solid phase process, it can be purified by a sequence of chromatographic steps including any or all of the following: ion exchange on a weakly basic resin in an acetate form; gel permeation chromatography, e.g., Sephadex G-25 columns; hydrophobic adsorption chromatography on underivatized polystyrene-divinyl benzene (for example, Amberlite XAD) ; silica gel absorption chromatography; ion exchange chromatography, e.g., on Sephadex G-25, or countercurrent distribution, or high-performance liquid chromatography (HPLC) , preferably reverse-phase HPLC on an octyl- or octadecylisilyl- silica bonded phase column.

Although the MB-40 peptide can be synthetically produced by the above-mentioned chemical methods, especially the solid-phase process, with high efficiency, it is also within the scope of this invention to produce the MB-40 peptide and fragments or analogs thereof, by genetic engineering technology. Thus, the DNA sequence encoding the desired polypeptide can be isolated or chemically synthesized, cloned into an appropriate bacterial plasmid and transformed into bacteria such as E. coli, which will then express the cloned sequence, resulting in production of the desired polypeptide. Once the desired polypeptide is purified it may be used to stimulate the release of IL-lα in vitro or in vivo, preferably in vivo, as IL-1 has a relatively short half-life when purified. Peptide MB-40 will be potentially useful either individually or in combination with other treatments for patients with abnormal immune defenses or following bone marrow transplantation.

In normal human thymus, there are three major cellular elements: thymocytes, epithelial cells and macrophages. It is believed that these three types of cells interact with each other to accomplish "thymic education." It has been reported that thymus epithelial cells may secrete hormones that cause the cortical thymocytes to differentiate into medullar thymocytes. Gatz, et al., 1982, Biol Resp. Cancer, 1, 219; Goldstein, et al., 1979, Science, 204, 1309, Dardenne, et al. , 1980 Clin. Exp. Immunol. 42, 477. It has further been suggested that thymocyte maturation may be triggered by the binding observed to occur between thymic macrophages and immature thymocytes. Lipsky and Rosenthal, 1973, J. Exp. Med. 138, 900; Lopez, et al., 1977, J. of Immunol. 119, 1668; Agrwal and Thomas, 1984, Cell Immunol. 84, 352. Thus, a functional linkage between thymic epithelial cells, macrophages and thymocytes is suggested.

The thymic epithelial cells may secrete thymic hormones such as thymosin B-₄ and thymosin α_lf as well as other thymic factors, such as MB-40. The thymic hormones may then act on thymocytes to induce their differentiation and other thymic factors may stimulate the macrophages to produce IL-lα and mIL-1, for example. IL-1 may then induce T-cell differentiation and maturation in the thymus and contribute to potentiating the self-antigen presentation to thymocytes and help to establish self/non-self discrimination. It follows that during the aging process, the involution of the thymus gland and the consequent decreased thymic hormone activity may result in macrophage imbalances, faulty antigen presentation and lack of adequate self-recognition. IL-1 is an important mediator of the body's responses to bacterial invasion, inflammatory processes and other immunologic reactions. IL-1 has also been shown to protect mice from the lethal effects of radiation and it enhances the proliferation of human and murine primitive non-cycling hematopoietic stem cells.

The biological properties and activities of IL-lα suggest that the peptide of this invention, MB-40, can be used clinically to induce IL-1 production and thus modulate the immune response. For example, MB-40 or its biologically active analogs can be administered to patients either alone or in combination with other therapies, in order to combat life- threatening diseases, such as cancer or infectious diseases that result in immune imbalances. Induction of endogenous IL- lα production by MB-40 may induce T-cell differentiation and maturation in the thymus and contribute to potentiating the self-antigen presentation to thymocytes and help to establish self/non-self discrimination.

The ability to stimulate immune system functions is known to be therapeutically useful in treating diseases such as infectious diseases caused by bacteria, viruses or fungi, conditions in which the immune system is deficient due to acquired or congenital conditions such as DiGeorge Syndrome, a congenital condition in which the thymus is absent, cancer and other afflictions of humans or animals. These peptides may be useful in the clinical treatment of conditions in which immunologic competence is believed to play an important role, for example, autoimmune diseases (e.g., lupus erythematosus, autoimmune hemolytic anemia, thyrotoxicoses) , thymic aplasia and dysplasia, decrease in immunologic competence due to a temporal decline in thymic hormone production, in chemical or radiologically induced im uno-suppressed states, etc. The immuno stimulating activity of MB-40 and its biological analogs can be applied to such diseases and conditions listed above as well as to other conditions not listed. Such immuno stimulation is also useful to boost the body's protective antibody response to such substances as vaccines. The peptides of this invention may be administered in combination with such vaccines.

Peptide MB-40 also has inhibitory effects upon the immune system in that the peptide inhibits the release of IL-6, an important mediator of immune responses. This inhibitory property of MB-40 can be used clinically to inhibit the release of IL-6 from such cells as anterior pituitary cells or medial basal hypothalami and thus modulate the immune response. For example, MB-40 or its biologically active analogs can be administered to patients, either alone or in combination with other therapies in order to reduce the concentration of IL-6 in the peripheral circulation. Effects of such treatment would be a reduction in the release of pituitary hormones such as growth hormone, OCTH, PRL and LH as well as a reduction in the synthesis of acute phase proteins in hepatocytes during inflammatory responses. As such, peptide MB-40 offers a useful tool in controlling the effects of an over-active immune response.

MB-40 and its biologically active analogs also modulate the effects of age on the graft versus host disease response. Aging induces a number of changes in the immune system, including the involution of the thymus and the consequent loss of thymic hormone production. Studies indicate that age of the recipient is an important prognostic factor for the development of either a chronic or acute host versus graft disease reaction following bone marrow transplantation. For example, the age of donor mice affects the graft versus host disease response of the donor's lymphocytes and administration of peptide MB-40 modulates that effect. When donor mice are injected with peptide MB-40 prior to transplantation of their spleen cells into recipient mice, a marked decrease in target cell lysis and an increase in immunoglobulin production are observed in the recipient mice. These symptoms are consistent with a chronic graft versus host disease response whereas untreated donor mice spleen cells caused an acute graft versus host disease response in recipient mice, characterized by low immunoglobulin production and high target cell lysis. Thus, it appears that aging, which results in a loss of thymic hormone production, may lead to an acute graft versus host disease response which can be modulated by administration of MB-40.

Another mechanism whereby peptides of the present invention may stimulate immune responsiveness involves modulation of the growth factor or mechanism that is responsible for regulating age-related alterations in lymphocytes. Aging results in loss of thymic hormone production and an apparent alteration in lymphocytes. Peptide MB-40 and its biologically active analogs appear to revert the lymphocytes to their original phenotype and activity, presumably by indirectly activating a lymphocyte modulating mechanism.

Pharmaceutical compositions that include the peptides of this invention as an active ingredient include the peptide MB-40 of formula I or an analog thereof containing from 8 to 60, preferably 10 to 50 amino acids, or a non-toxic salt of any of these dispersed in a pharmaceutically acceptable carrier. Also included is the peptide of formula I or an analog thereof, covalently linked at its carboxyl terminus to the amino terminus of the peptide of formula II or an analog thereof containing from 25-50 amino acids, or a non-toxic salt of any of these, dispersed as above. The amount of peptide in the composition can range from about 0.1 to 99% by weight of the composition. Appropriate dosages of the peptides of the invention will depend upon the condition presented by the individual subject. The skilled medical worker will be able to determine appropriate dosages based upon the circulating levels of IL-lα required to combat the physiological disability. However, in general, amounts of from about 1 mg to about 100 mg/kg body weight/day of the biologically active peptide should be useful. In view of the above, it should be noted that the present invention comprises methods of treatment that comprise administration of therapeutically effective doses of the peptides of this invention to patients in need of same. The peptides may be mixed with a variety of carrier compounds depending on the form of preparation desired for administration, e.g., oral, nasal, rectal or parenteral. The dosage will be determined for the route of administration.

In preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be used, such as water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents in the case of oral liquid, preparations such as suspension, elixirs or solutions.

In the case of oral solid preparations, such as powders, capsules and tablets, typical carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents may be employed. If desired, tablets may be sugar coated or enteric coated by standard techniques. For parenterals, the carrier will usually comprise sterile water, although other inert ingredients may be added to aid in solubility or for preservative purposes. Injectable suspensions may be prepared, in which case the appropriate liquid carriers and suspending agents may be added. Compositions of sublingual, rectal or nasal administration may also be prepared with any of the usual pharmaceutical excipients.

Example 1 - Isolation of MB-40

Fractions of TF5 were purified by reversed-phase chromatography on a Delta-prep HPLC system equipped with a Model 481 variable wave length detector with a semi-preparative flow-cell, set at 280 η , and 300 x 500 mm Delta-pak, 300 A, 15 μm C₁₈ column (Waters). Eluent A was 0.02M ammonium acetate (pH 6.8) and eluent B was aceto-nitrile. A 60-min linear gradient from 0-80% B was run at a flow-rate of 80 ml/min. TF5 was dissolved in the initial buffer and applied to the column. One minute fractions were collected and combined into seven different pools referred to as MB1-MB7 in order of hydrophobicity. See Fig. 2. Each of the seven pooled fractions was incubated with normal human peripheral blood monocytes at dosages of 500 μg/ml,

100 μg/ml, 10 μg/ml and 1 μg/ml and assayed for the stimulation of IL-1 production. The IL-1 assay is described in Example 2. The results are shown in Table 1. Fractions MB1-MB5 showed no significant increase of IL-1 production above background levels. Fraction MB6 and especially MB7 showed a significant IL-1 inducing capacity. MB7 appeared to be the more potent IL- 1 stimulating fraction in that 1.0 μg/ml of this fraction stimulated a significant increase in IL-1 production.

Further analysis of fractions 41-57 in pool MB 7 indicate that the IL-1 stimulating peptide elutes in fraction 50 (F50) . F50 contained the peak of activity and therefore was used for further purification.

F50 was further fractionated on a 150 x 3.9 mm-I.D. Delta-pak 300 A 5 m C₁₈ column using model 510 HPLC system (Waters) , equipped with a Model 441 detector set at 214 n . Eluent A was 0.1% phosphoric acid in water and eluent B was acetonitrile with 0.1% phosphoric acid. A 60 min. linear gradient from 0-35% B with 10 minute hold was run at a flow rate of 1 ml/min. One-half minute fractions were collected and assayed for IL-1 activity. The results of the RP-HPLC of F50 are shown in Figure 3. Peptide MB-40 elutes in fraction 60 (F60) , the peak of IL-1 activity. F60 was concentrated to remove the acetonitrile and re-chromatographed under the same conditions. The results are shown in Figure 4. The peak (peak l in Figure 4) was collected and identified as peptide MB-40 by iso-electricfocusing gel electrophoresis (IEF) , SDS-poly¬ acrylamide gel electrophoresis (SDS-PAGE) , amino acid composition and sequence analysis.

Isoelectric focusing analysis of MB-40 using LKB/Pharmacia isoelectric focusing gels, pH 3.5-9.5 run at constant power was performed. The gels were fixed and stained in 20% trichloracetic acid and Coomassie Blue R250 and destained in acetic acid, ethanol and water (1.3.8, v/v) . The results are shown in Figure 5. In Lane 1 10 μg LKB/Pharmacia IEF pH markers, pH 3-10, were separated; Lane 2 contains 20 μg MB-40; Lane 3 contains 75 μg thymosin fraction 5 and Lane 4 contains 10 μg ubiquitin.

SDS-PAGE of peptide MB-40 was performed using a 1.5 nm 16% SDS-polyacrylamide gel according to the method of Laemmli (1970) and stained using Coomassie Blue. The results are shown in Figure 6. Lane 1 contains 1 ml low molecular weight markers from Biorad and Lane 2 shows the migration of 2 μg of peptide MB-40. MB-40 migrated as a single band with a molecular weight of approximately 4-5 kiloDaltons.

Amino acid analysis was performed with the Pico-Tag amino acid analysis system of Waters-Millipore. The method is based on the formation of a phenyl thiocarbamyl (PTC) derivative of each amino acid from acid-hydrolyzed proteins.

Peptide MB-40 samples (around 1-5 μg) were hydrolyzed in 200

1 of a constant boiling HC1 atmosphere containing 1% (v/v) phenol, at 110°C for 24, 48, 72 and 120 hours in a Pico-Tag work station. The hydrolysates were dried and the amino acids were derivatized with phenylisothiocyanate (PITC) for 20 minutes at room temperature to yield the corresponding phenylthiocarbamyl derivatives. These derivatives were analyzed with the Pico-Tag amino acid analysis system, which had previously been calibrated with a standard of amino acids.

Table 2 shows the assumed number of residues per molecule based on these analysis. The number of residues based upon sequence analysis is also given in this table.

Amino acid sequence analysis was performed on a Beckman 890 M Sequencer using Beckman standard operating program 52285. The sequencer products were identified using model 510 HPLC system (Waters) equipped with Model 441 detector set at 254 nm. The peptide sequence derivatives, phenylthiohydantion (PTH) amino acids were reconstituted in 10 ml of acetonitrile and 2 ml was injected to the 150 x 3.9 mm Nova-Pak C₁₈ column. Eluent A was 25 mM Na-acetate, pH 5.0 in acetonitrile (5.5:lv/v) and eluent B was 2-propanol in water (3:2v/v). The column temperature was held constant at 40°C. The gradient conditions were as follows: GRADIENT TABLE

Carboxyl-terminal sequence determination of peptides was performed with carboxyl-peptidase Y (CPY) . The peptide (0.2-1 nmole) was dissolved in 49 ml of 50 mM Na-acetate buffer, pH 5.5 and 500 ng of CPY in 1 ml deionized water was added and incubated at room temperature. At various time intervals, T=l, 2, 5, 10, 20, 30 and 60 minutes, 5 ml were removed and the reaction was immediately stopped by the addition of 5 ml glacial acetic acid to the aliquot. The samples were freeze-dried and submitted to amino acid analysis using a Pico-Tag amino acid analysis system.

Table 2

*The data are presented as numbers of residues per molecule, ^•aspartic acid and glutamic acid values are the sum of their acids and amines.

Example 2 - IL-1 Assay Human peripheral blood lymphoid cells are obtained from heparinized blood of normal volunteers by separation of the mononuclear cells on Ficoll-hypaque (Flow Laboratories, McLean, Virginia) . The cells are washed three times and placed onto plastic petri dishes for 2 hours at 37°C. After 2 hours incubation, the non-adherent cells are washed off and the remaining cells are scraped off using a rubber policeman, resulting in a population of peripheral blood monocytes. The cells are washed and resuspended at a concentration of 2 x 10⁶ cells/ml of Click's EHAA medium (Irvine Scientific, Santa Ana, California) supplemented with 25 mM HEPES buffer, 10% FCS, 2mM L-glutamine, 5 x 10^~5 M 2-mercaptoethanol and penicillin- streptomycin (100 units and 100 μg/ml, respectively) . 100 ml of the cell suspension are seeded onto microliter culture wells and incubated at 37°C for 2 hours. At the end of the incubation, the wells are washed to remove any non-adherent cells and 0.1 ml of fresh culture medium is applied to the wells. Incubation of 2 x 10⁵ of the double adherent human peripheral blood monocytes with different doses of TF5, ubiquitin and peptide MB-40 is carried out for 24 hours at 37°C. The culture plates are centrifuged prior to collection of the supernatants to remove any cells. The culture supernatant is assayed for the presence of IL-1 using DI0.G4.1 cells (Kaye, et al., 1984 J. Immuno. 133_r 1399). 2 x 10⁴ DI0.G4.1 cells are cultured in Click's medium in the presence of 1 μg/ml concanavalin A and serial dilutions of the supernatants at 37°C for 72 hours. Six hours prior to harvesting, the cells are pulse labelled with 1 m Ci of ³H-thymidine (DuPont, Boston, Massachusetts) . All determinations were done in triplicate and the results are presented as units/ml (representative of 1 x 10⁶ macrophages) using a standard curve prepared with purified human IL-1 (Genzyne Corp. , Boston, Massachusetts) .

Incubation of human peripheral blood monocytes with different doses of TF5, ubiquitin and peptide MB-40 results in a significant increase in the production of IL-lα. Peptide MB-40 is significantly more potent than the partially purified TF5 in production of IL-1 and ubiquitin and MB-40 stimulate induction to the same extent. The results are shown in Figure 7.

Example 3 - Chemical Synthesis of Peptide MB-40 Met-Gln-Ile-Phe-Val-Lys-Thr-Leu-Thr-Gly-Lys-Thr-Ile-Glu-Leu- Glu-Val-Glu-Pro-Ser-Asp-Thr-Ile-Glu-Asn-Val-Lys-Ala-Lys-Ile- Gln-Asp-Lys-Ser-Gly-Ile-Pro-Pro-Asp-Gln-NH₂.

A. p-Methylbenzhydrylmine Resin (1.08 g; 0.50 mmole) is placed in a 150 ml peptide synthesis flask, washed three times with dichloromethane (20 ml, 2 min each) , one time with 10% triethylamine in dichloromethane, stirred 10 min in 10% triethylamine in dichloromethane followed by three washings with dichloromethane. The neutralized resin is stirred with Boc-Gln (0.37g; 1.5 mmol), 1-hydroxybenzotriazole (0.46 g; 3 mmol) and N,N*-dicyclohexylcarbodiimide (0.309 g; 1.5 mmol) for 180 min in 20 ml N,N-dimethylformamide. The Boc-Gln-Resin thus formed is subjected to the following steps in each solid phase peptide synthesis cycles (20 volumes of solvent or reagent: 1.5 mmol each of suitably protected Boc-amino acid and N,N- dicyclohexcarbodiimide were used in each step unless otherwise stated) : (1) prewash with 50% trifluoroacetic acid in dichloromethane,

(2) stir 30 minutes in 50% trifluoroacetic acid in dichloromethane,

(3) wash 3 times with dichloromethane, (4) prewash with 10% triethylamine in dichloromethane,

(5) stir 3 minutes in 10% triethylamine in dichloromethane ,

(6) test for ninhydrin reaction (should be strongly positive) ,

(7) add 0.48 g Boc-Asp (OBzl) and 0.309 g N,N'- dicyclohexyl carbodiimide in 12 ml of dichloromethane and stir for 120 minutes,

(8) wash 2 times with 50% isopropyl alcohol in dichloromethane,

(9) wash 3 times with dichloromethane,

(10) test for ninhydrin reaction (should be negative at this point, if not, repeat steps 7-10) .

The synthetic cycle was repeated using the following Boc-amino acid (each 15 mmol) , sequentially, one at a time in step 7:

Boc-Pro, Boc-Pro, Boc-Ile, Boc-Gyl, Boc-Ser(Bzl) , Boc-Lys(Clz) , Boc-

Asp(OBzl) , Boc-Gln, Boc-Ile, Boc- Lys(Clz), Boc-Ala, Boc-Lys (Clz) , Boc-Val, Boc-Asn, Boc Glu(OBzl) , Boc-Ile, Boc-Thr(Bzl) , Boc- Asp(OBzl), Boc-Ser(Bzl) , Boc-Pro,

Boc-Glu(OBzl) , Boc-Val, Boc- Glu(OBzl), Boc-Leu, Boc-Glu(OBzl) , Boc-Ile, Boc-Thr(Bzl) , Boc-Lys(Clz) , Boc-Gly, Boc-Thr(Bzl) , Boc-Leu, Boc- Thr(Bzl), Boc-Lys(Clz) , Boc-Val,

Boc-Phe, Boc-Ile, Boc-Gln and Boc- Met. For any synthetic cycle involving Boc-Asn or Boc-Gln in step 7, the reaction takes place in N,N-dimethylformamide (12 volumes) for 180 minutes in the presence of 3 mmol of 1- hydroxybenzotriazole and 1.5 mmol N,N'-dicyclohexyl- carbondiimide.

Upon completion of the synthesis, 4.13 g of protected peptide resin, Boc-Met-Gln-Ile-Phe-Val-Lys(Czl)- Thr(Bzl)-Leu-Thr(Bzl)-Gly-Lys(Czl)-Thr(Bzl)-Ile-Glu(OBzl)-Leu-

Glu(OBzyl)-Val-Glu(OBzl)-Pro-Ser(Bzl)-Asp(OBzl)-Thr(Bzl)-Ile-

Glu(OBzl)-Asn-Val-Lys(Clz)-Ala-Lys(Clz)-Ile-Gln-Asp(OBzl)-

Lys(Clz)-Ser(Bzl)-Gly-Ile-Pro-Pro-Asp(OBzl)-Gln-HN-CH(-C₆H₄-

Me)-C₆H₄-Resin, was obtained. Part of this protected peptide resin (1.5 g) was mixed with 3 ml of anisole and treated with approximately 25 ml of anhydrous liquid HF at 0°C for 45 minutes. Excess acid was removed under vacuum and the residue was thoroughly washed with anhydrous ether, resulting in a slightly off white colored powder. The peptide material was then extracted from the powder with 50 ml of 2% ammonium acetate solution which on desalting by a Sephadex G-10 column (2.6 x 85 cm; 0.1 M HOAC) and lyophilization provided by 0.47g of crude peptide MB-40. B. Purification of Synthetic Peptide MB-40 Crude peptide MB-40 from part A was purified on a

150 x 3.9 mm-I.D. Delta-A 5 μm C₁₈ column using model 510 HPLC system (Waters) , equipped with model 441 detector set at 214 nm. Eluent A was 20 mM potassium phosphate buffer, pH 6.0 and Eluent B was acetoritrite with 50% Eluent A. Separation of synthetic peptide MB-40 was achieved with a 40 minute linear gradient from 0-40% B. Results indicated that synthetic peptide MB-40 elutes in Fraction 29 (F29) , as shown in Figure 8. F29 was concentrated to remove acetonitrile and rechromotographed under the same conditions as shown in Figure 9. The peak was collected and subjected to IEF (Figure 10), SDS-PAGE (Figure 11) and amino acid composition analysis (Table 5).

Example 4 - Biological Activities of Peptide MB-40

A. Using the IL-1 assay described in Example 2, human peripheral blood monocytes are incubated with 1 g,

500 μg, 50 μg, 10 μg, 0.5 μg, and 0.05 μg/ml MB-40 or TF5. The results are shown in Figure 12.

Synthetic peptide MB-40 is significantly more potent than the crude TF5 extract in production of IL-lα. Incubation of peripheral blood monocytes with MB-40 elicited a concentration dependent stimulation of IL-lα production, with a maximum stimulatory response at 100 μg/ml MB-40 in this assay.

B. This example demonstrates the modulating effects of MB-40 on age-induced changes in the induction of graft-versus-host disease by DBA/21 mouse lymphocytes. Four month-old DBA/21 female mice were injected with various doses of MB-40 (μg/day dilute in PBS containing 1% BSA) for four days and then the mice were sacrificed and their spleen cells removed by lavage. These spleen cells were then injected into B6D2F_X mice. Fourteen days later the spleen cells from the F₂ mice were obtained and tested for the presence of CTL by measuring the lysis of ⁵¹Cr-labelled EL4 cells (150:1 effector to target ratio) and for the production of immunoglobulin relative to a normal F_± control using an ELISA assay. Spleen cells from untreated 4 month old DBA/21 mice induce an acute form of GVHD which is characterized by high CTL (% specific lysis of EL 4 + target cells) and low immunoglobulin production. Spleen cells from control untreated young DBA/21 mice induce chronic GVHD characterized by low or no CTL and high immunoglobulin production. The doses of MB-40 tested were 1.0, 0.1, 0.01, 0.001 and 0.0001 μg/day. The results are shown in Figure 13. Peptide MB-40-treated spleen cells cause a poor GVHD response in recipient mice, indicated by low GTL and low antibody production. These preliminary results suggest that peptide MB-40 may prove clinically useful in pre-treating bone marrow donors or cells prior to bone marrow transplants in order to reduce GVHD response.

C. This example demonstrates the inhibitory effects of MB-40 on I/L-6 release from the medial basal hypothalamus (MBH) . Medial basal hypothalami were removed from the brains of Sprague-Dawley adult rats and divided into quarters by sagittal and coronal sections centered about the median eminence. The sections were randomly placed in wells of 24-well tissue culture plates along with 500 1 Krebs-Ringer buffer containing 0.025% BSA. Preincubation was carried out for 90 minutes in a 95% 0₂ - 5% C0₂ atmosphere, after which time the medium was removed and the MBH were exposed to 0.1 - 1.0 μg/ml peptide MB-40 in basal medium for a 90 minute incubation period. MBH controls were incubated in basal medium lacking peptide MB-40. The conditioned media were sterilized and stored at 4°C pending IL-6 bioassay.

The concentration of IL-6 that accumulated in the incubation medium of the MBH was quantitated using IL-6 dependent 7TD1 hybridoma bioassay. Aliquots of the MBH/MB-40 conditioned medium and MBH-conditioned medium of the control samples were serially diluted two-fold in RPMI-1640 medium in microtiter plates in duplicate, after which 2 X 10³ 7TD1 cells/well were added and cultured for 3 days in RPMI-1640 medium supplemented with 5% fetal calf-serum, 5 μg/ml gentamycin and 50 μm 2-mercaptoethanol. Three dilutions of each sample were routinely included in each assay. The proliferation of 7TD1 cells was determined calorimetrically. The IL-6 concentration of each of the samples was determined by comparison to the linear portion of a standard curve generated with rmIL-6.

The presence of MB-40 had no effect on the proliferation of 7TD1 cells in the absence or presence of rmlL- 6. The results are shown in Figure 14. The release of IL-6 by MBH is inhibited by peptide MB-40 by 50-60% in a concentration dependent manner. At a concentration of 0.01 μg/ml, MB-40 inhibits the release of IL-6 by 60%, whereas higher concentrations of MB-40 have a decreased inhibitory effect.

Claims

What Is Claimed Is: Claim 1. A peptide having the ability to induce the production of interleukin 1-α (IL-lα) from human peripheral blood monocytes, said peptide having from about 10 to 50 amino acids and including the whole or part of the peptide having the amino acid sequence represented by the formula: Met-Gln-Ile-Phe-Val-Lys-Thr-Leu-Thr- Gly-Lys-Thr-Ile-X-_L-Leu-Glu-Val-Glu- Pro-Ser-Asp-Thr-Ile-Glu-Asn-Val-Lys- Ala-Lys-Ile-Gln-Asp-Lys-X₂-Gly-Ile- Pro-Pro-Asp-Gin wherein X_λ is Glu or Thr and X₂ is Ser or Glu or an analog thereof having all or part of the primary structure and biological activity of the peptide of said sequence. Claim 2. A peptide having the ability to induce the production of IL-lα from human peripheral blood monocytes, said peptide having from about 10 to 50 amino acids and including the whole or part of the peptide having the amino acid represented by the formula: Met-Gln-Ile-Phe-Val-Lys-Thr-Leu-Thr- Gly-Lys-Thr-Ile-Thr-Leu-Glu-Val-Glu- Pro-Ser-Asp-Thr-Ile-Glu-Asn-Val-Lys- Ala-Lys-Ile-Gln-Asp-Lys-Glu-Gly-Ile- Pro-Pro-Asp-Gin. Claim 3. A peptide having the formula: Met-Gln-Ile-Phe-Val-Lys-Thr-Leu-Thr-Gly-Lys-Thr-Ile-X^Leu-Glu- Val-Glu-Pro-Ser-Asp-Thr-Ile-Glu-Asn-Val-Lys-Ala-Lys-Ile-Gln- Asp-Lys-X -Gly-Ile-Pro-Pro-Asp-Gln. Claim 4. A peptide having the formula: Met-Gln-Ile-Phe-Val-Lys-Thr-Leu-Thr-Gly-Lys-Thr-Ile-Thr-Leu- Glu-Val-Glu-Pro-Ser-Asp-Thr-Ile-Glu-Asn-Val-Lys-Ala-Lys-Ile- Gln-Asp-Lys-Glu-Gly-Ile-Pro-Pro-Asp-Gl . Claim 5. A method for stimulating the in vitro release of Interleukin-lα from human peripheral blood monocytes that comprises incubating the monocytes in cell culture medium containing a peptide having the composition set forth in Claim 1. Claim 6. A method for stimulating the in vitro release of Interleukin-la from human peripheral blood monocytes that comprises incubating the monocytes in cell culture medium containing a peptide having the composition set forth in Claim 2. Claim 7. A method for the clinical treatment of conditions in which immunological competence is decreased such as, autoimmune diseases or chemically or radiologically induced immuno-suppression comprising administering an effective amount of the peptide of Claim 1 to a patient in need of such treatment. Claim 8. The method of Claim 7 which comprises administering said peptide and other thymic hormone stimulant other than said peptide. Claim 9. A method for treating graft rejection after bone marrow or organ transplantation comprising administering an effective amount of the peptide of Claim 1 to a patient in need of such treatment. Claim 10. The method of Claim 9 which comprises administering said peptide and other thymic hormone stimulant other than said peptide. Claim 11. A pharmaceutical composition for stimulating the release of interleukin lα comprising an effective amount of the peptide of Claim 1 and a pharmaceutically acceptable solid or liquid carrier. Claim 12. The pharmaceutical composition of Claim 11 further comprising an effective amount of another thymic hormone stimulant other than said peptide. Claim 13. A method for inhibiting the in vitro release of Interleukin-6 from basal hypothalami and anterior pituitary cells that comprises incubating said cells in culture medium containing a peptide having the composition as set forth in claim 1. Claim 14. A method for inhibiting the in vitro release of Interleukin-6 from medial basal hypothalami and anterior pituitary cells that comprises incubating said cells in culture medium containing a peptide having the composition as set forth in claim 2.