WO2004069270A1 - Immunoregulating compounds and an associated method - Google Patents

Immunoregulating compounds and an associated method Download PDF

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Publication number
WO2004069270A1
WO2004069270A1 PCT/US2003/002627 US0302627W WO2004069270A1 WO 2004069270 A1 WO2004069270 A1 WO 2004069270A1 US 0302627 W US0302627 W US 0302627W WO 2004069270 A1 WO2004069270 A1 WO 2004069270A1
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Prior art keywords
glu
lys
group
amino
asp
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PCT/US2003/002627
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French (fr)
Inventor
Lili Guo
Roger W. Roeske
Wei Li
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Advanced Research And Technology Institute, Inc.
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Priority to AU2003210714A priority Critical patent/AU2003210714A1/en
Priority to CN038262037A priority patent/CN1819839B/en
Priority to PCT/US2003/002627 priority patent/WO2004069270A1/en
Publication of WO2004069270A1 publication Critical patent/WO2004069270A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/57581—Thymosin; Related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present disclosure generally relates to immunoregulating compounds and an associated method.
  • the present disclosure particularly relates to compounds possessing biological activity similar to thymosin.
  • the thymus gland is an important central lymphoid organ in the neck or upper thorax of all mammals.
  • the thymus gland is most prominent during early life. In many laboratory species of mammals and in humans it reaches its greatest relative weight at the time of birth, but its absolute weight continues to increase until the onset of puberty. Thereafter, it begins to undergo an involution and progressively decreases in size throughout adult life.
  • the thymic stem cells generate a large population of small lymphocytes (thymocytes) through a series of mitotic divisions. Simultaneously these dividing lymphocytes show evidence of cellular differentiation within the special thymic environment.
  • T lymphocytes undergo an intrathymic migration from the peripheral cortical area to the medullary core of the organ. Some thymocytes degenerate within the organ, but many enter the circulating blood and lymph systems at various stages of maturity. A small percentage of the T lymphocyte population (5-10%) within the thymus is antigenically competent and capable of recognizing antigenic sites on foreign cells or substances. Some of the T lymphocytes have the capacity to lyse the foreign tissue cells, while others are involved in recognizing the "foreignness" of the antigens and assisting a second sub-population of bone-marrow-derived lymphocytes (B lymphocytes) to respond to the antigen by producing a specific antibody. These two types of immunocompetent T lymphocytes are called killer cells and helper cells, respectively. They are involved in both tissue transplantation and humoral antibody responses.
  • Thymosin is a polypeptide hormone synthesized and secreted by the endodermally derived reticular cells of the thymus gland. Thymosin exerts its actions in several loci: (1) in the thymus gland, either on precursor stem cells derived from fetal liver or from bone marrow, or on immature thymocytes, and (2) in peripheral sites, on either thymic-derived lymphoid cells or on precursor stem cells.
  • the precursor stem cells which are immunologically incompetent whether in the thymus or in peripheral sites, have been designated as predetermined T cells or T 0 cells, and mature through stages termed Ti and T 2 each reflecting varying degrees of immunological competence.
  • Thymosin promotes or accelerates the maturation of T 0 cells to Ti cells as well as to the final stage of a T 2 .
  • the hormone also increases the number of total lymphoid cells by accelerating the rate of proliferation of both immature and mature lymphocytes.
  • Thymosin has significant therapeutic value as a result of its above described ability to stimulate lymphocyte production. For example, it has been reported that thymosin has been clinically successful in preventing and treating primary and secondary infections, modulating the deleterious effects of radiotherapy and chemotherapy, accelerating the rate of wound healing, decreasing some of the effects of aging, and improving the efficacy of other treatments. In addition, there are reports that thymosin has been successfully utilized as an adjuvant in surgery and treatments using antifungal, antibiotic, and antiviral agents (e.g. utilized in the treatment of hepatitis B and hepatitis C).
  • thymosin possesses desirable biological activity. Accordingly, compounds possessing biological activity similar to thymosin are desirable.
  • composition in accordance with the present disclosure comprises one or more of the features or combinations thereof:
  • a compound having the amino acid sequence Ser-Asp-Ala-Ala-Val-Asp- Tl r-Ser-Ser-Glu-Ile-Tlir-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala- Glu-Asn (SEQ ID NO:l)
  • amino acid sequence X-Ser-Asp- Ala-Ala- Val-Asp-Thr- Ser-Ser-Glu-Ile-Tl r-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu- Asn (SEQ ID NO:2)
  • X is an acetyl group or H, or a variant of these amino acid sequences that is at least about 70% homologous to the amino acid sequence.
  • At least one peptide bond between amino acids of the amino acid sequences or variant thereof is formed between a pair of functional groups selected from the group consisting of ⁇ - carboxy group of Asp and ⁇ -amino group of Ala, ⁇ -carboxy group of Asp and ⁇ -amino group of Thr, ⁇ -carboxy group of Asp and ⁇ -amino group of Leu, ⁇ -carboxy group of Glu and ⁇ -amino group of He, ⁇ -carboxy group of Glu and ⁇ -amino group of Lys, ⁇ -carboxy group of Glu and ⁇ -amino group of Lys, ⁇ -carboxy group of Glu and ⁇ -amino group of Lys, ⁇ -carboxy group of Glu and ⁇ -amino group of Lys, ⁇ -carboxy group of Glu and ⁇ -amino group of Val, ⁇ -carboxy group of Glu and ⁇ - amino group of Glu, ⁇ -car
  • the compound can be included in a composition having a pharmaceutically acceptable carrier.
  • the compound can have activity in an E-Rosette forming or inhibition assay or lymphocyte proliferation test, or mixed lymphocyte reaction.
  • the compound can be utilized in a method to reconstitute an immune function of an animal, such as a mammal.
  • FIG. 1 is a HPLC elutogram of thymosin alpha 1 (retention time 27.56 min.) and a compound of the present disclosure (retention time 26.04 min.);
  • FIG. 2 A is a LCQ electrospray mass spectra of thymosin alpha 1
  • FIG. 2B is a LCQ electrospray mass spectra of the compound of the present disclosure having a retention time of 26.04 min. in the elutogram shown in FIG. i
  • FIG. 3 is a graph depicting the effect of various concentrations of the compound of the present disclosure having a retention time of 26.04 min. in the elutogram shown in FIG. 1 on a rosette-forming capacity (RFC) test as compared to the same concentration of thymosin alpha 1 on the RFC test
  • FIG. 4 is a graph illustrating the rosette-forming activities of 7 compounds of the present disclosure and thymosin alpha 1 at the same concentration;
  • FIG. 5 is a schematic representation of an exemplary polypeptide of the present disclosure
  • FIG. 6 is a schematic representation of another exemplary polypeptide of the present disclosure
  • FIG. 7 is still another schematic representation of an exemplary polypeptide of the present disclosure.
  • FIG. 8 is yet another schematic representation of an exemplary polypeptide of the present disclosure.
  • FIG. 9 is still another schematic representation of an exemplary polypeptide of the present disclosure.
  • FIG. 10 is yet another schematic representation of an exemplary polypeptide of the present disclosure
  • FIG. 11 is still another schematic representation of an exemplary polypeptide of the present disclosure.
  • thymosin alpha 1 or thymosin is a 28 amino acid polypeptide having desirable immunomodulatory properties which is synthesized and secreted by the endodermally derived reticular cells of the thymus gland.
  • Thymosin alpha 1 has the following amino acid sequence:
  • each peptide bond in the thymosin alpha 1 molecule is formed between an ⁇ -amino functional group of one amino acid in the amino acid sequence and the ⁇ -carboxy functional group of the adjacent amino acid in the amino acid sequence.
  • the present disclosure is directed to compositions or compounds which have similar biological activity as thymosin alpha 1 but, as discussed in greater detail below, have a distinct chemical structure.
  • the present disclosure is directed to amino acid sequences similar to the aforementioned amino acid sequence with one difference being that at least one peptide bond is not formed between an ⁇ -amino functional group of one amino acid and an ⁇ -carboxy functional group of another amino acid.
  • the present disclosure is directed to compounds which have 70% or greater homology with the aformentioned amino acid sequence and have one or more peptide bonds formed between a pair of functional groups selected from the group consisting of ⁇ -carboxy group of Asp and ⁇ -amino group of Ala, ⁇ -carboxy group of Asp and ⁇ -amino group of Thr, ⁇ -carboxy group of Asp and ⁇ -amino group of Leu, ⁇ -carboxy group of Glu and ⁇ -amino group of He, ⁇ -carboxy group of Glu and ⁇ -amino group of Lys, ⁇ -carboxy group of Glu and ⁇ -amino group of Lys, ⁇ -carboxy group of Glu and ⁇ -amino group of Lys, ⁇ -carboxy group of Glu and ⁇ -amino group of Lys, ⁇ -carboxy group of Glu and ⁇ -amino group of Val, ⁇ -carboxy group of
  • homologous refers to the identity of amino acids included in the sequence and does not include structural isomers of the sequence.
  • polypeptide is used in the broadest sense to mean a sequence of amino acids that can be encoded by a cellular gene, or by recombinant nucleic acid sequence, or can be chemically synthesized.
  • polypeptides of the present disclosure can be chemically synthesized in a substantially purified form that is relatively free from contaminating lipids, proteins, nucleic acids or other cellular material normally associated with a polypeptide in a cell.
  • the polypeptides described herein can be a portion of a larger polypeptide or be the entire polypeptide.
  • polypeptides described herein have similar biological activity as thymosin alpha 1 and in one embodiment include polypeptides having an amino acid sequence as follows: X-Ser-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 (SEQ ID NO:2) or a variant of the amino acid sequence that is (i) at least about 70% homologous to the amino acid sequence and (ii) have activity (e.g.
  • X is an acetyl group or H and (iii) at least one peptide bond between amino acids of the amino acid sequence or variant thereof is formed between a pair of functional groups selected from the group consisting of ⁇ - carboxy group of Asp and ⁇ -amino group of Ala, ⁇ -carboxy group of Asp and ⁇ -amino group of Thr, ⁇ -carboxy group of Asp and ⁇ -amino group of Leu, ⁇ -carboxy group of Glu and ⁇ -amino group of He, ⁇ -carboxy group of Glu and ⁇ -amino group of Lys, ⁇ -carboxy group of Glu and ⁇ -amino group of Lys, ⁇ -carboxy group of Glu and ⁇ -amino group of Lys, ⁇ -carboxy group of Glu and ⁇ -amino group of Lys, ⁇ -carboxy group of Glu and ⁇ -amino group of Val, ⁇ -carboxy group
  • polypeptides of the present disclosure are identical to those described above with the exception that variants of the amino acid sequence are least about 75% homologous to the amino acid sequence X-Ser-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 (SEQ ID NO:2).
  • polypeptides of the present disclosure are identical to those described above with the exception that variants of the amino acid sequence are least about 80% homologous to the amino acid sequence X-Ser-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 (SEQ ID NO:2).
  • polypeptides of the present disclosure are identical to those described above with the exception that variants of the amino acid sequence are least about 85% homologous to the amino acid sequence X-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-GTu- Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn (SEQ ID NO:2).
  • polypeptides of the present disclosure are identical to those described above with the exception that variants of the amino acid sequence are least about 90%> homologous to the amino acid sequence X-Ser-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 (SEQ ID NO:2).
  • polypeptides of the present disclosure are identical to those described above with the exception that variants of the amino acid sequence are least about 95% homologous to the amino acid sequence X-Ser- 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 (SEQ ID NO :2).
  • polypeptide of the present disclosure is a polypeptide having an amino acid sequence as follows: Ac-Ser-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 (SEQ ID NO:3), where, as indicated in FIG.
  • a peptide bond is formed between the ⁇ -carboxy group of Asp and the ⁇ -amino group of Ala
  • a peptide bond is formed between the ⁇ -carboxy group of Asp and the ⁇ -amino group of Thr
  • a peptide bond is formed between the ⁇ -carboxy group of Asp and ⁇ -amino group of Leu (hereinafter referred to as polypeptide ⁇ -Asp 2 ' 6 ' 15 ) .
  • polypeptide of the present disclosure is one having an amino acid sequence as follows: Ac-Ser-Asp-Ala-Ala-Val-Asp-Tl ⁇ r-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys- Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn (SEQ ID NO:3), where, as shown in FIG. 6, a peptide bond is formed between the ⁇ -carboxy group of Asp and the ⁇ -amino group of Ala (hereinafter referred to as polypeptide ⁇ -Asp 2 ).
  • polypeptide of the present disclosure is one having an amino acid sequence as follows: Ac-Ser-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 (SEQ ID NO:3), where, as illustrated in FIG. 7, a peptide bond is formed between the ⁇ -carboxy group of Asp and the ⁇ -amino group of Thr (hereinafter referred to as polypeptide ⁇ -Asp ).
  • polypeptide of the present disclosure is one having an amino acid sequence as follows: Ac-Ser-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 (SEQ ID NO:3), where, as depicted in FIG. 8, a peptide bond is formed between the ⁇ -carboxy group of Asp and ⁇ -amino group of Leu (hereinafter referred to as polypeptide ⁇ -Asp 15 ).
  • polypeptide of the present disclosure is one having an amino acid sequence as follows: Ac-Ser- Asp- Ala-Ala- Val- Asp-Thr-Ser-Ser-Glu-Ile-Thr-Tlir-Lys-Asp-Leu-Lys-Glu-Lys- Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn (SEQ ID NO:3), where, as shown in FIG.
  • a peptide bond is formed between the ⁇ -carboxy group of Asp and the ⁇ -amino group of Ala and a peptide bond is formed between the ⁇ -carboxy group of Asp and the ⁇ -amino group of Thr (hereinafter referred to as polypeptide ⁇ -Asp 2,6 ).
  • polypeptide of the present disclosure is one having an amino acid sequence as follows: Ac-Ser-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 (SEQ ID NO:3), where, as illustrated in FIG.
  • a peptide bond is formed between the ⁇ -carboxy group of Asp and the ⁇ -amino group of Ala and a peptide bond is formed between the ⁇ -carboxy group of Asp and ⁇ -amino group of Leu (hereinafter referred to as polypeptide ⁇ -Asp 2 ' 15 ).
  • polypeptide of the present disclosure is one having an amino acid sequence as follows: Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile- Tl r-Tl r-Lys-A ⁇ p-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn (SEQ ID NO:3), where, as illustrated in FIG.
  • polypeptide ⁇ -Asp 6,15 a peptide bond is formed between the ⁇ -carboxy group of Asp and the ⁇ -amino group of Thr and a peptide bond is formed between the ⁇ - carboxy group of Asp and ⁇ -amino group of Leu (hereinafter referred to as polypeptide ⁇ -Asp 6,15 ).
  • Polypeptides of the present disclosure can be synthesized utilizing solid phase chemistry in conjunction with an appropriate blocking group.
  • polypeptides of the present disclosure can be synthesized using the solid phase method on an Applied Biosystems automated synthesizer (ABI, model 431 A, which is commercially available from Applied Biosystems Inc.
  • Fmoc 9- fluorenylmethoxycarbonyl
  • Trityl Wang resin(p-Benzyloxybenzyl Alcohol resin, 0.40 mmol/g, which is commercially available from Novabiochem located in San Diego, California, 0.25 millimole scale, with "FastMoc" Procedure.
  • Each cartridge contained 1.0 millimole of the appropriate Fmoc amino acid.
  • the resin was filtered off and the peptide was precipitated by addition of 350 ml cold ether.
  • the precipitate was dissolved in 100 ml of 20% acetonitrile in water/1.0M NH 4 OAc (20:80v/v), diluted to 300 ml with water and lyophilized.
  • the crude peptide was purified by reversed phase HPLC on a C18 column (Vydac 218TP 101550). Fractions that were pure (analytical HPLC) were combined and lyophilized. The molecular mass was verified by electrospray mass spectroscopy.
  • each polypeptide ⁇ - Asp 2 ' 6 ' 15 , ⁇ -Asp 2 , ⁇ -Asp 6 , ⁇ -Asp 15 , ⁇ -Asp 2 ' 6 , ⁇ -Asp 2 ' 15 , and ⁇ -Asp 6 ' 15 is set forth below.
  • cartridges containing one millimole (a 4-fold excess) of each protected amino acid were loaded into the above described automated synthesizer apparatus (i.e. ABI, model 431 A).
  • the ⁇ -amino group of each amino acid was protected by an Fmoc group.
  • the side chain functional groups were protected as follows: ⁇ -carboxyls of glutamic acid residues 10, 18, 21, 24, 25 and 27 as the t-butyl ester; the ⁇ -amino groups of lysines 14, 17, 19 and 20 as the t-BOC (t-butyloxycarbonyl) group; the ⁇ -carboxyls of Asp 2 ' 6 ' 15 as the t-butyl esters, allowing only the ⁇ -carboxyl of Asp to become activated by HBTU and HOBt.
  • the hydroxyl side chains of Ser 1 ' 8 ' 9 and Thr 7,'12,'13 were protected by t-butyl ether formation.
  • Polypeptides ⁇ -Asp 2 , ⁇ -Asp 6 , ⁇ -Asp 15 , ⁇ -Asp 2 ' 6 , ⁇ -Asp 2 ' 15 and ⁇ -Asp 6 ' 15 were synthesized in the same way, the only variable being the placement of the side chain protecting groups on the Aspartic acid residues as illustrated below in Table 1.
  • the HPLC elutogram of the comparison of polypeptide ⁇ -Asp ' ' with thymosin alpha 1 is shown in FIG. 1. Retention time of the polypeptide ⁇ -Asp ' ' (26.04 min) is lower than thymosin alpha 1 (27.56 min) which correlates with better solubility.
  • the conditions for the analytical HPLC were as follows: solvent A: 0.1 % TFA in water; B: 70% CH 3 CN/H 2 O, 0.09% TFA. The elution was run with a 0-20% B gradient over 30 min at a flow rate of 1.0 ml/min on a Vydac C 18 4.6 x 250 mm column.
  • the synthetic yield of the polypeptide ⁇ -Asp 2 ' 6 ' 15 is 26%, which is higher than thymosin alpha 1 (19 %).
  • the MS spectrum of polypeptide ⁇ -Asp 2 ' 6 ' 15 is illustrated in FIG. 2B while that of thymosin alpha 1 is shown in FIG. 2 A.
  • Theoretical values of MH + , MH 2 ++ , MH 3 + and MH 4 are 3107.5, 1554.3, 1036.5 and 777.6 respectively for each compound.
  • the functional activity and /or therapeutically effective dose of the polypeptides of the present disclosure can be assayed in vitro, by various methods. These methods are based on the physiological processes involved in immunoregulation and while they are within the scope of the present disclosure, they are not intended to limit the methods by which the polypeptides of the present disclosure are defined and/or therapeutically effective dosage of the polypeptides are determined.
  • several biological systems have been used to establish the activity of immunomodulators, such as thymosins, in vitro.
  • One technique known as rosette-cell formation has been a sensitive and effective method.
  • the E-Rosette Assay was used to evaluate the activity of polypeptides ⁇ -Asp 2 ' 6 ' 15 , ⁇ -Asp 2 , ⁇ -Asp 6 , ⁇ -Asp 15 , ⁇ -Asp 2 ' 6 , ⁇ -Asp 2 ' 15 , and ⁇ -Asp 6 ' 15 . .
  • Heparin solution 200 units Heparin/ml isotonic sodium chloride solution.
  • Alserver's solution NaCl (0.42g), glucose (2g), citric acid monohydrate (0.055g) and sodium citrate dihydrate (0.766g) were dissolved in 100 ml water and sterilized by boiling for one hour.
  • Stain solution mix 2 ml Gimesa stain solution with 6 ml hanks, centrifuge at 1500 rpm/min for 10 min, and then take supernatant.
  • Gimesa stain solution 0.5g Gimesa dye was added to 33 ml glycerol, which is warmed to 55-60 C° to have the dye dissolved. 33 ml methanol is added at room temperature (RT) and allowed to stand at RT for 24 hours. Filter and keep the filtrate as stock solution.
  • o Pig thymus thymus is freshly taken from a healthy pig.
  • Sheep blood sheep venous blood was collected and treated with the anticoagulant
  • the fresh pig thymus glands were cut into small pieces after the fat was removed.
  • Appropriate Hanks buffer was added and the suspension was filtered through 100- aperture sieve.
  • the supernatant was transferred into a centrifuge tube, which contains one-third volume of lymphocyte separating solution and centrifuged for 20 minutes at 2000 rpm.
  • the interface cells were collected, suspended in appropriate Hank's solution and centrifuged for 3 minutes at 1500 rpm, which is repeated three times.
  • the final lymphocyte concentration was made to 4x10 cells/ml Hanks.
  • Sheep red blood cells were washed three times as were T lymphocytes and made to a final concentration of 4x10 7 cells/ml Hanks.
  • Thymosin alpha 1 and ⁇ -Asp 2 ' 6 ' 15 were dissolved in Hanks in serials of 200 ⁇ g/ml,
  • ⁇ -Asp 2 , ⁇ -Asp 6 , ⁇ -Asp 15 , ⁇ -Asp 2 ' 6 , ⁇ -Asp 2 ' 15 , and ⁇ -Asp 6 ' 15 were made to 200 ⁇ g/ml in Hanks.
  • 0.1 ml of peptide sample was added to a tube and 0.1 ml of Hank's to the control.
  • 0.2 ml of 4x10 6 cells/ml of T lymphocyte was added and incubated for one hour at 37 C°.
  • 0.2 ml of 4x10 7 cells/ml of SRBC was added and mixed gently. The mixture was centrifuged for three minutes at 500 rpm and left at 4° overnight. After the supernatant was removed the cells were suspended by gentle shaldng and stained with Gimesa stain. More than 200 T cells were counted in each slide to calculate RFC%(rosette forming cell).
  • FIG. 3 The result of total erythrocyte-rosette-forming test of ⁇ -Asp 2 ' 6 ' 15 and thymosin alpha 1 is shown in FIG. 3.
  • the percentage of RFC increased significantly from 16%o to 35%) when 0.1 ml of ⁇ -Asp 2 ' ' 5 or thymosin alpha 1 was added at 0.02 ⁇ g/ml or higher concentration after the subtraction of the effect of the control. No significant difference was observed between ⁇ -Asp ' ' and thymosin alpha 1.
  • FIG. 3 The result of total erythrocyte-rosette-forming test of ⁇ -Asp 2 ' 6 ' 15 and thymosin alpha 1 is shown in FIG. 3.
  • the percentage of RFC increased significantly from 16%o to 35%) when 0.1 ml of ⁇ -Asp 2 ' ' 5 or thymosin alpha 1 was added at 0.02 ⁇ g/ml or higher concentration after the subtraction of the
  • ⁇ -Asp 2 ' 6 ' 15 shows rosette-forming activities of polypeptides ⁇ -Asp 2 ' 6 ' 15 (indicated as sample 8 on the x-axis), ⁇ -Asp 2 (indicated as sample 1 on the x-axis), ⁇ -Asp 6 (indicated as sample 2 on the x-axis), ⁇ -Asp 15 (indicated as sample 3 on the x-axis), ⁇ -Asp 2 ' 6 (indicated as sample 4 on the x-axis), ⁇ -Asp 2 ' 15 (indicated as sample 5 on the x-axis), and ⁇ -Asp 6 ' 15 (indicated as sample 6 on the x-axis) as compared with thymosin alpha 1 (indicated as sample 7 on the x-axis).
  • polypeptides of the present disclosure have significant therapeutic value as a result of their immunomodulatory effects.
  • polypeptides of the present disclosure can be utilized in treating primary and secondary infections, modulating the deleterious effects of radiotherapy and chemotherapy, accelerating the rate of wound healing, decreasing some of the effects of aging, and improving the efficacy of other treatments.
  • the polypeptides of the present disclosure may be utilized as an adjuvant in surgery and treatments using antifungal, antibiotic, and antiviral agents (e.g. utilized in the treatment of hepatitis B and hepatitis C).
  • the therapeutically effective dosage for the enhancement of immune functions in vivo may be extrapolated from in vitro assays (e.g. E-Rosette Assay) using the polypeptides and/or compositions of the present disclosure alone or in combination with other immunoregulating factors.
  • in vitro assays e.g. E-Rosette Assay
  • the compounds or polypeptides of the present disclosure can be utilized in a, for example, immunomodulating or immunoregulating composition.
  • the polypeptides of the present disclosure can be utilized in a immunopotentiating composition for reconstituting an immune function in a mammal.
  • a method for reconstituting an immune function in a mammal can comprise administering to the mammal an immunopotentiating effective amount of a compound or polypeptide having an amino acid sequence as follows: X-Ser-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 (SEQ ID NO:2) or a variant of the amino acid sequence that is (i) at least about 70% or more (e.g.
  • X is an acetyl group or H and (ii) at least one peptide bond between amino acids of the amino acid sequence or variant thereof is formed between a pair of functional groups selected from the group consisting of ⁇ -carboxy group of Asp and ⁇ -amino group of Ala, ⁇ -carboxy group of Asp and ⁇ -amino group of Thr, ⁇ -carboxy group of Asp and ⁇ -amino group of Leu, ⁇ -carboxy group of Glu and ⁇ -amino group of He, ⁇ -carboxy group of Glu and ⁇ -amino group of Lys, ⁇ -carboxy group of Glu and ⁇ -amino group of Lys, ⁇ -carboxy group of Glu and ⁇ -amino group of Lys, ⁇ -carboxy group of Glu and ⁇ -amino group of Lys, ⁇ -carboxy group of Glu and ⁇ -amino group of Lys, ⁇ -carboxy
  • the present disclosure also provides pharmaceutical compositions.
  • the pharmaceutical compositions of the present disclosure can be administered to any animal which may experience the beneficial effects of the compounds of the disclosure. Foremost among such animals are humans, although the disclosure is not intended to be so limited.
  • the pharmaceutical compositions of the invention can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
  • compositions of the present disclosure may be administered by any method that achieves their intended purpose.
  • administration may be by parenteral , subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or buccal routes.
  • administration may be by the oral route.
  • the dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
  • the term "pharmaceutically acceptable” means approved by a regulatory agency of a Federal or state government, or listed in the U.S. Pharmacopeia or other generally recognized Pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerine, glycol or other synthetic solvents. Water can be utilized as a carrier when the pharmaceutical composition is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates.
  • Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for the adjustment of tonicity such as sodium chloride or dextrose are also contemplated.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose made of glass or plastic.
  • compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides, microcrystalline cellulose, gum tragacanth or gelatin.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.
  • Such compositions will contain a therapeutically effective amount of a therapeutic (e.g. a polypeptide of the present disclosure), preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
  • the formulation should suit the mode of administration.
  • the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to an animal, such as a human being.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • compositions of the present disclosure are those large enough to produce the desired effect, whereby, for example, an immunopotentiating effect is achieved.
  • the doses should not be so large as to cause adverse side effects, such as unwanted cross reactions anaphalactic reactions and the like.
  • the dosage will vary with the age, condition, sex and extent of the disease or condition in the patient . Counterindication, if any, immune tolerance and other variables will also affect the proper dosage.
  • the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner in view of the particular circumstances.

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Abstract

Immunoregulating compounds and an associated method.

Description

4.
IMMTJNOREGULATING COMPOUNDS AND AN ASSOCIATED METHOD
TECHNICAL FIELD OF THE DISCLOSURE
The present disclosure generally relates to immunoregulating compounds and an associated method. The present disclosure particularly relates to compounds possessing biological activity similar to thymosin.
BACKGROUND OFTHEDISCLOSURE
The thymus gland is an important central lymphoid organ in the neck or upper thorax of all mammals. The thymus gland is most prominent during early life. In many laboratory species of mammals and in humans it reaches its greatest relative weight at the time of birth, but its absolute weight continues to increase until the onset of puberty. Thereafter, it begins to undergo an involution and progressively decreases in size throughout adult life. The thymic stem cells generate a large population of small lymphocytes (thymocytes) through a series of mitotic divisions. Simultaneously these dividing lymphocytes show evidence of cellular differentiation within the special thymic environment. During this division and maturation phase the developing thymocytes undergo an intrathymic migration from the peripheral cortical area to the medullary core of the organ. Some thymocytes degenerate within the organ, but many enter the circulating blood and lymph systems at various stages of maturity. A small percentage of the T lymphocyte population (5-10%) within the thymus is antigenically competent and capable of recognizing antigenic sites on foreign cells or substances. Some of the T lymphocytes have the capacity to lyse the foreign tissue cells, while others are involved in recognizing the "foreignness" of the antigens and assisting a second sub-population of bone-marrow-derived lymphocytes (B lymphocytes) to respond to the antigen by producing a specific antibody. These two types of immunocompetent T lymphocytes are called killer cells and helper cells, respectively. They are involved in both tissue transplantation and humoral antibody responses.
Thymosin (thymosin alpha 1) is a polypeptide hormone synthesized and secreted by the endodermally derived reticular cells of the thymus gland. Thymosin exerts its actions in several loci: (1) in the thymus gland, either on precursor stem cells derived from fetal liver or from bone marrow, or on immature thymocytes, and (2) in peripheral sites, on either thymic-derived lymphoid cells or on precursor stem cells. The precursor stem cells, which are immunologically incompetent whether in the thymus or in peripheral sites, have been designated as predetermined T cells or T0 cells, and mature through stages termed Ti and T2 each reflecting varying degrees of immunological competence. Thymosin promotes or accelerates the maturation of T0 cells to Ti cells as well as to the final stage of a T2. In addition to this maturation influence, the hormone also increases the number of total lymphoid cells by accelerating the rate of proliferation of both immature and mature lymphocytes. Thymosin has significant therapeutic value as a result of its above described ability to stimulate lymphocyte production. For example, it has been reported that thymosin has been clinically successful in preventing and treating primary and secondary infections, modulating the deleterious effects of radiotherapy and chemotherapy, accelerating the rate of wound healing, decreasing some of the effects of aging, and improving the efficacy of other treatments. In addition, there are reports that thymosin has been successfully utilized as an adjuvant in surgery and treatments using antifungal, antibiotic, and antiviral agents (e.g. utilized in the treatment of hepatitis B and hepatitis C).
In light of the above discussion it is apparent that thymosin possesses desirable biological activity. Accordingly, compounds possessing biological activity similar to thymosin are desirable.
SUMMARY OF THE DISCLOSURE
A composition in accordance with the present disclosure comprises one or more of the features or combinations thereof:
A compound having the amino acid sequence Ser-Asp-Ala-Ala-Val-Asp- Tl r-Ser-Ser-Glu-Ile-Tlir-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala- Glu-Asn (SEQ ID NO:l), or the amino acid sequence X-Ser-Asp- Ala-Ala- Val-Asp-Thr- Ser-Ser-Glu-Ile-Tl r-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu- Asn (SEQ ID NO:2)where X is an acetyl group or H, or a variant of these amino acid sequences that is at least about 70% homologous to the amino acid sequence. At least one peptide bond between amino acids of the amino acid sequences or variant thereof is formed between a pair of functional groups selected from the group consisting of β- carboxy group of Asp and α-amino group of Ala, β-carboxy group of Asp and α-amino group of Thr, β-carboxy group of Asp and α-amino group of Leu, γ-carboxy group of Glu and α-amino group of He, α-carboxy group of Glu and ε-amino group of Lys, γ-carboxy group of Glu and α-amino group of Lys, γ-carboxy group of Glu and ε-amino group of Lys, γ-carboxy group of Glu and α-amino group of Val, γ-carboxy group of Glu and α- amino group of Glu, γ-carboxy group of Glu and α-amino group of Ala, γ-carboxy group of Glu and α-amino group of Asn, α-carboxy of Thr and ε-amino of Lys, α-carboxy of Leu and ε-amino of Lys, α-carboxy of Lys and ε-amino of Lys, carboxy group of β-Ala and α-amino group of Ala, carboxy group of β-Ala and α-amino group of Glu, and carboxy group of β-Ala and α-amino group of Val. The compound can be included in a composition having a pharmaceutically acceptable carrier. The compound can have activity in an E-Rosette forming or inhibition assay or lymphocyte proliferation test, or mixed lymphocyte reaction. The compound can be utilized in a method to reconstitute an immune function of an animal, such as a mammal.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a HPLC elutogram of thymosin alpha 1 (retention time 27.56 min.) and a compound of the present disclosure (retention time 26.04 min.);
FIG. 2 A is a LCQ electrospray mass spectra of thymosin alpha 1; FIG. 2B is a LCQ electrospray mass spectra of the compound of the present disclosure having a retention time of 26.04 min. in the elutogram shown in FIG. i; FIG. 3 is a graph depicting the effect of various concentrations of the compound of the present disclosure having a retention time of 26.04 min. in the elutogram shown in FIG. 1 on a rosette-forming capacity (RFC) test as compared to the same concentration of thymosin alpha 1 on the RFC test; FIG. 4 is a graph illustrating the rosette-forming activities of 7 compounds of the present disclosure and thymosin alpha 1 at the same concentration;
FIG. 5 is a schematic representation of an exemplary polypeptide of the present disclosure; FIG. 6 is a schematic representation of another exemplary polypeptide of the present disclosure;
FIG. 7 is still another schematic representation of an exemplary polypeptide of the present disclosure;
FIG. 8 is yet another schematic representation of an exemplary polypeptide of the present disclosure;
FIG. 9 is still another schematic representation of an exemplary polypeptide of the present disclosure;
FIG. 10 is yet another schematic representation of an exemplary polypeptide of the present disclosure; and FIG. 11 is still another schematic representation of an exemplary polypeptide of the present disclosure.
DETAILED DESCRIPTION OF THE DISCLOSURE
While the disclosure is susceptible to various modifications and alternative forms, a specific embodiment thereof has been shown by way of example and will herein be described in detail. It should be understood, however, that there is no intent to limit the disclosure to the particular form disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the disclosure as defined by the appended claims. As discussed above, thymosin alpha 1 or thymosin is a 28 amino acid polypeptide having desirable immunomodulatory properties which is synthesized and secreted by the endodermally derived reticular cells of the thymus gland. Thymosin alpha 1 has the following amino acid sequence:
Ac-Ser-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 (SEQ ID NO:3) It should be appreciated that each peptide bond in the thymosin alpha 1 molecule is formed between an α-amino functional group of one amino acid in the amino acid sequence and the α-carboxy functional group of the adjacent amino acid in the amino acid sequence. The present disclosure is directed to compositions or compounds which have similar biological activity as thymosin alpha 1 but, as discussed in greater detail below, have a distinct chemical structure. For example, in one embodiment the present disclosure is directed to amino acid sequences similar to the aforementioned amino acid sequence with one difference being that at least one peptide bond is not formed between an α-amino functional group of one amino acid and an α-carboxy functional group of another amino acid. For example, in one embodiment, the present disclosure is directed to compounds which have 70% or greater homology with the aformentioned amino acid sequence and have one or more peptide bonds formed between a pair of functional groups selected from the group consisting of β-carboxy group of Asp and α-amino group of Ala, β-carboxy group of Asp and α-amino group of Thr, β-carboxy group of Asp and α-amino group of Leu, γ-carboxy group of Glu and α-amino group of He, α-carboxy group of Glu and ε-amino group of Lys, γ-carboxy group of Glu and α-amino group of Lys, γ-carboxy group of Glu and ε-amino group of Lys, γ-carboxy group of Glu and α-amino group of Val, γ-carboxy group of Glu and α-amino group of Glu, γ-carboxy group of Glu and α- amino group of Ala, γ-carboxy group of Glu and α-amino group of Asn, α-carboxy of Thr and ε-amino of Lys, α-carboxy of Leu and ε-amino of Lys, α-carboxy of Lys and ε-amino of Lys, carboxy group of β-Ala and α-amino group of Ala, carboxy group of β-Ala and α- amino group of Glu, and carboxy group of β-Ala and α-amino group of Val.
Note that as used herein "homology" refers to the identity of amino acids included in the sequence and does not include structural isomers of the sequence. For example, the amino acid sequence Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr- Thι--Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn (SEQ ID NO:l) where a peptide bond is formed between the β-carboxy group of Asp and α-amino group of Ala would still be 100% homologous with the amino acid sequence Ser- Asp-Ala- Ala- Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thι--Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu- Glu-Ala-Glu-Asn (SEQ ID NO:l) where a peptide bond is formed between the β-carboxy group of Asp and α-amino group of Thr.
It should be appreciated that, as used herein, the term "polypeptide" is used in the broadest sense to mean a sequence of amino acids that can be encoded by a cellular gene, or by recombinant nucleic acid sequence, or can be chemically synthesized. For example, polypeptides of the present disclosure can be chemically synthesized in a substantially purified form that is relatively free from contaminating lipids, proteins, nucleic acids or other cellular material normally associated with a polypeptide in a cell. In addition, it is contemplated that the polypeptides described herein can be a portion of a larger polypeptide or be the entire polypeptide.
As mentioned above, the polypeptides described herein have similar biological activity as thymosin alpha 1 and in one embodiment include polypeptides having an amino acid sequence as follows: X-Ser-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 (SEQ ID NO:2) or a variant of the amino acid sequence that is (i) at least about 70% homologous to the amino acid sequence and (ii) have activity (e.g. immunopotentiating activity) in an E-Rosette forming assay, where X is an acetyl group or H and (iii) at least one peptide bond between amino acids of the amino acid sequence or variant thereof is formed between a pair of functional groups selected from the group consisting of β- carboxy group of Asp and α-amino group of Ala, β-carboxy group of Asp and α-amino group of Thr, β-carboxy group of Asp and α-amino group of Leu, γ-carboxy group of Glu and α-amino group of He, α-carboxy group of Glu and ε-amino group of Lys, γ-carboxy group of Glu and α-amino group of Lys, γ-carboxy group of Glu and ε-amino group of Lys, γ-carboxy group of Glu and α-amino group of Val, γ-carboxy group of Glu and α- amino group of Glu, γ-carboxy group of Glu and α-amino group of Ala, γ-carboxy group of Glu and α-amino group of Asn, α-carboxy of Thr and ε-amino of Lys, α-carboxy of Leu and ε-amino of Lys, α-carboxy of Lys and ε-amino of Lys, carboxy group of β-Ala and α-amino group of Ala, carboxy group of β-Ala and α-amino group of Glu, and carboxy group of β-Ala and α-amino group of Val. In another embodiment, the polypeptides of the present disclosure are identical to those described above with the exception that variants of the amino acid sequence are least about 75% homologous to the amino acid sequence X-Ser-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 (SEQ ID NO:2). In still another embodiment, the polypeptides of the present disclosure are identical to those described above with the exception that variants of the amino acid sequence are least about 80% homologous to the amino acid sequence X-Ser-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 (SEQ ID NO:2). In yet another embodiment, the polypeptides of the present disclosure are identical to those described above with the exception that variants of the amino acid sequence are least about 85% homologous to the amino acid sequence X-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-GTu- Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn (SEQ ID NO:2). In a further embodiment, the polypeptides of the present disclosure are identical to those described above with the exception that variants of the amino acid sequence are least about 90%> homologous to the amino acid sequence X-Ser-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 (SEQ ID NO:2). In still another embodiment, the polypeptides of the present disclosure are identical to those described above with the exception that variants of the amino acid sequence are least about 95% homologous to the amino acid sequence X-Ser- 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 (SEQ ID NO :2).
One specific example of a polypeptide of the present disclosure is a polypeptide having an amino acid sequence as follows: Ac-Ser-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 (SEQ ID NO:3), where, as indicated in FIG. 5, a peptide bond is formed between the β-carboxy group of Asp and the α-amino group of Ala, a peptide bond is formed between the β-carboxy group of Asp and the α-amino group of Thr, and a peptide bond is formed between the β-carboxy group of Asp and α-amino group of Leu (hereinafter referred to as polypeptide β-Asp2'6'15) . Another specific example of a polypeptide of the present disclosure is one having an amino acid sequence as follows: Ac-Ser-Asp-Ala-Ala-Val-Asp-Tlτr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys- Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn (SEQ ID NO:3), where, as shown in FIG. 6, a peptide bond is formed between the β-carboxy group of Asp and the α-amino group of Ala (hereinafter referred to as polypeptide β-Asp2). Still another specific example of a polypeptide of the present disclosure is one having an amino acid sequence as follows: Ac-Ser-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 (SEQ ID NO:3), where, as illustrated in FIG. 7, a peptide bond is formed between the β-carboxy group of Asp and the α-amino group of Thr (hereinafter referred to as polypeptide β-Asp ). Yet another specific example of a polypeptide of the present disclosure is one having an amino acid sequence as follows: Ac-Ser-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 (SEQ ID NO:3), where, as depicted in FIG. 8, a peptide bond is formed between the β-carboxy group of Asp and α-amino group of Leu (hereinafter referred to as polypeptide β-Asp15). Still another specific example of a polypeptide of the present disclosure is one having an amino acid sequence as follows: Ac-Ser- Asp- Ala-Ala- Val- Asp-Thr-Ser-Ser-Glu-Ile-Thr-Tlir-Lys-Asp-Leu-Lys-Glu-Lys- Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn (SEQ ID NO:3), where, as shown in FIG. 9, a peptide bond is formed between the β-carboxy group of Asp and the α-amino group of Ala and a peptide bond is formed between the β-carboxy group of Asp and the α-amino group of Thr (hereinafter referred to as polypeptide β-Asp2,6). Still another specific example of a polypeptide of the present disclosure is one having an amino acid sequence as follows: Ac-Ser-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 (SEQ ID NO:3), where, as illustrated in FIG. 10, a peptide bond is formed between the β-carboxy group of Asp and the α-amino group of Ala and a peptide bond is formed between the β-carboxy group of Asp and α-amino group of Leu (hereinafter referred to as polypeptide β-Asp2'15). Another specific example of a polypeptide of the present disclosure is one having an amino acid sequence as follows: Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile- Tl r-Tl r-Lys-A§p-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn (SEQ ID NO:3), where, as illustrated in FIG. 11, a peptide bond is formed between the β-carboxy group of Asp and the α-amino group of Thr and a peptide bond is formed between the β- carboxy group of Asp and α-amino group of Leu (hereinafter referred to as polypeptide β-Asp6,15). Polypeptides of the present disclosure can be synthesized utilizing solid phase chemistry in conjunction with an appropriate blocking group. For example, polypeptides of the present disclosure can be synthesized using the solid phase method on an Applied Biosystems automated synthesizer (ABI, model 431 A, which is commercially available from Applied Biosystems Inc. located in Foster City, California, using Fmoc (9- fluorenylmethoxycarbonyl)-t-butyl chemistry. A run is started with Fmoc-Asn(Trityl) Wang resin(p-Benzyloxybenzyl Alcohol resin, 0.40 mmol/g, which is commercially available from Novabiochem located in San Diego, California, 0.25 millimole scale, with "FastMoc" Procedure. Each cartridge contained 1.0 millimole of the appropriate Fmoc amino acid. Coupling was carried out by HBTU [2-(lH-benzotriazol-l-yl)-l, 1,3,3- tetramethyluronium hexafluorophosphate] and HOBt (1-Hydroxybenzotriazole ) and the Fmoc protecting group was removed by 20 % piperidine in NMP(N-methylpyrrolidone) after each coupling. When the assembly of amino acids was complete, the peptide was cleaved from resin by 20 ml of a mixture of TFA (Trifluoroacetic Acid), phenol, EDT (1,2-ethanedithiol) and water(20:l:0.5:l) at room temperature for two hours. The resin was filtered off and the peptide was precipitated by addition of 350 ml cold ether. The precipitate was dissolved in 100 ml of 20% acetonitrile in water/1.0M NH4OAc (20:80v/v), diluted to 300 ml with water and lyophilized. The crude peptide was purified by reversed phase HPLC on a C18 column (Vydac 218TP 101550). Fractions that were pure (analytical HPLC) were combined and lyophilized. The molecular mass was verified by electrospray mass spectroscopy.
A more detailed description of the synthesis of each polypeptide β- Asp2'6'15, β-Asp2, β-Asp6, β-Asp15, β-Asp2'6, β-Asp2'15, and β-Asp6'15 is set forth below. In the synthesis of the polypeptide β-Asp2'6'15 , cartridges containing one millimole (a 4-fold excess) of each protected amino acid were loaded into the above described automated synthesizer apparatus (i.e. ABI, model 431 A). The α-amino group of each amino acid was protected by an Fmoc group. The side chain functional groups were protected as follows: γ-carboxyls of glutamic acid residues 10, 18, 21, 24, 25 and 27 as the t-butyl ester; the ε-amino groups of lysines 14, 17, 19 and 20 as the t-BOC (t-butyloxycarbonyl) group; the α-carboxyls of Asp2'6'15 as the t-butyl esters, allowing only the β-carboxyl of Asp to become activated by HBTU and HOBt. The hydroxyl side chains of Ser1'8'9 and Thr 7,'12,'13 were protected by t-butyl ether formation. The remaining 8 residues - Val 5,20,23 Leu16, He11 and Ala3'24'26 required no side chain protection. The contents of the amino acid cartridges were dissolved and delivered to the activation vessel, then to the reaction vessel by pressure of nitrogen gas. The synthesis proceeded from C-terminal to N- terminal. Disconnection of the assembled polypeptide from the polystyrene resin was achieved as previously discussed.
Polypeptides β-Asp2, β-Asp6, β-Asp15, β-Asp2'6, β-Asp2'15 and β-Asp6'15 were synthesized in the same way, the only variable being the placement of the side chain protecting groups on the Aspartic acid residues as illustrated below in Table 1.
TABLE 1
Figure imgf000011_0001
As indicated above, the molecular mass of each polypeptide β-Asp2' '15, β-Asp2, β-Asp6, β-Asp15, β-Asp2'6, β-Asp2'15, and β-Asp6'15 was verified by electrospray mass spectroscopy. The synthetic yield and mass spectrometric analysis (MW) for each of the polypeptides β-Asp2'6'15, β-Asp2, β-Asp6, β-Asp15, β-Asp2'6, β-Asp2'15, and β-Asp6'15 was -l i¬
as follows: β-Asp2'6'15 synthetic yield was 26.0%>, mass spectrometric analysis showed that the compound had the expected molecular weight; MH+=3,108.0(Calculated MW=3,107.5); β-Asp2 synthetic yield was 10.3%, mass spectrometric analysis showed that the compound had the expected molecular weight; MH+=3,108.6(Calculated MW=3,107.5); β-Asp6 synthetic yield was 8.0%>, mass spectrometric analysis showed that the compound had the expected molecular weight; MH+=3,108.9(Calculated MW=3,107.5); β-Asp15 synthetic yield was 4.8%, mass spectrometric analysis showed that the compound had the expected molecular weight; MH+=3,108.4(Calculated MW=3,107.5), β-Asp2'6 synthetic yield was 15.8%), mass spectrometric analysis showed that the compound had the expected molecular weight; MH+=3 , 107.9(Calculated
MW=3,107.5); β-Asp2'15 synthetic yield is 5.5%, mass spectrometric analysis showed that the compound had the expected molecular weight; MH+=3,107.2(Calculated MW=3,107.5); and β-Asp6'15 synthetic yield was 10.1%, mass spectrometric analysis showed that the compound had the expected molecular weight; MH =3,108.6(Calculated MW=3,107.5).
The HPLC elutogram of the comparison of polypeptide β-Asp ' ' with thymosin alpha 1 is shown in FIG. 1. Retention time of the polypeptide β-Asp ' ' (26.04 min) is lower than thymosin alpha 1 (27.56 min) which correlates with better solubility. The conditions for the analytical HPLC were as follows: solvent A: 0.1 % TFA in water; B: 70% CH3CN/H2O, 0.09% TFA. The elution was run with a 0-20% B gradient over 30 min at a flow rate of 1.0 ml/min on a Vydac C18 4.6 x 250 mm column. Also note that the synthetic yield of the polypeptide β-Asp2'6'15 is 26%, which is higher than thymosin alpha 1 (19 %). The MS spectrum of polypeptide β-Asp2'6'15 is illustrated in FIG. 2B while that of thymosin alpha 1 is shown in FIG. 2 A. Theoretical values of MH+, MH2 ++, MH3 + and MH4 are 3107.5, 1554.3, 1036.5 and 777.6 respectively for each compound.
It should be appreciated that the functional activity and /or therapeutically effective dose of the polypeptides of the present disclosure can be assayed in vitro, by various methods. These methods are based on the physiological processes involved in immunoregulation and while they are within the scope of the present disclosure, they are not intended to limit the methods by which the polypeptides of the present disclosure are defined and/or therapeutically effective dosage of the polypeptides are determined. In particular, several biological systems have been used to establish the activity of immunomodulators, such as thymosins, in vitro. One technique known as rosette-cell formation has been a sensitive and effective method. Accordingly, as discussed below, the E-Rosette Assay was used to evaluate the activity of polypeptides β-Asp2'6'15, β-Asp2, β-Asp6, β-Asp15, β-Asp2'6, β-Asp2'15, and β-Asp6'15 ..
Reagents and Materials of the E-Rosette Assay:
• Heparin solution: 200 units Heparin/ml isotonic sodium chloride solution. • Alserver's solution: NaCl (0.42g), glucose (2g), citric acid monohydrate (0.055g) and sodium citrate dihydrate (0.766g) were dissolved in 100 ml water and sterilized by boiling for one hour.
• Fixing solution: 25% glutaraldehyde, 3.5% NaHCO3 and Hanks solution (1 : 1 :38).
• Hanks solution: Potassium dihydrogenphosphate (KH2PO , 0.06g), sodium hydrogenphosphate (Na2HPO4, 0.06g), KC1 0.4g, NaCl (8g) and glucose (lg) were dissolved in 1000 ml water. The pH was adjusted to 7.2-7.3 with 4%> Na2CO3 and sterilized.
• Stain solution: mix 2 ml Gimesa stain solution with 6 ml hanks, centrifuge at 1500 rpm/min for 10 min, and then take supernatant. • Gimesa stain solution: 0.5g Gimesa dye was added to 33 ml glycerol, which is warmed to 55-60 C° to have the dye dissolved. 33 ml methanol is added at room temperature (RT) and allowed to stand at RT for 24 hours. Filter and keep the filtrate as stock solution. o Pig thymus: thymus is freshly taken from a healthy pig. o Sheep blood: sheep venous blood was collected and treated with the anticoagulant
Heparin. Equal volume of Alserver's solution was added and stored at 4 C°. o Preparation of T-lymphocytes :
The fresh pig thymus glands were cut into small pieces after the fat was removed. Appropriate Hanks buffer was added and the suspension was filtered through 100- aperture sieve. The supernatant was transferred into a centrifuge tube, which contains one-third volume of lymphocyte separating solution and centrifuged for 20 minutes at 2000 rpm. The interface cells were collected, suspended in appropriate Hank's solution and centrifuged for 3 minutes at 1500 rpm, which is repeated three times. The final lymphocyte concentration was made to 4x10 cells/ml Hanks.
• Preparation of sheep red cells
Sheep red blood cells were washed three times as were T lymphocytes and made to a final concentration of 4x107 cells/ml Hanks.
• Preparation of peptide samples: Thymosin alpha 1 and β-Asp2'6'15 were dissolved in Hanks in serials of 200 μg/ml,
20 μg/ml, 2 μg/ml, 0.2 μg/ml and 0.02 μg/ml. β-Asp2, β-Asp6, β-Asp15, β-Asp2'6, β-Asp2'15, and β-Asp6'15 were made to 200 μg/ml in Hanks.
Experimental
0.1 ml of peptide sample was added to a tube and 0.1 ml of Hank's to the control. 0.2 ml of 4x106 cells/ml of T lymphocyte was added and incubated for one hour at 37 C°. Then 0.2 ml of 4x107 cells/ml of SRBC was added and mixed gently. The mixture was centrifuged for three minutes at 500 rpm and left at 4° overnight. After the supernatant was removed the cells were suspended by gentle shaldng and stained with Gimesa stain. More than 200 T cells were counted in each slide to calculate RFC%(rosette forming cell).
Results
The result of total erythrocyte-rosette-forming test of β-Asp2'6'15 and thymosin alpha 1 is shown in FIG. 3. The percentage of RFC increased significantly from 16%o to 35%) when 0.1 ml of β-Asp2' ' 5 or thymosin alpha 1 was added at 0.02 μg/ml or higher concentration after the subtraction of the effect of the control. No significant difference was observed between β-Asp ' ' and thymosin alpha 1. In a similar manner FIG. 4 shows rosette-forming activities of polypeptides β-Asp2'6'15 (indicated as sample 8 on the x-axis), β-Asp2 (indicated as sample 1 on the x-axis), β-Asp6 (indicated as sample 2 on the x-axis), β-Asp15 (indicated as sample 3 on the x-axis), β-Asp2'6 (indicated as sample 4 on the x-axis), β-Asp2'15 (indicated as sample 5 on the x-axis), and β-Asp6'15 (indicated as sample 6 on the x-axis) as compared with thymosin alpha 1 (indicated as sample 7 on the x-axis). There are no significant differences observed between thymosin alpha 1 and polypeptides β-Asp2' ' , β-Asp2, β-Asp6, β-Asp15, β-Asp2'6, β-Asp2'15, and β-Asp6'15 when 0.1 ml of samples were used at 200 μg/ml. Accordingly, the polypeptides of the present disclosure have significant therapeutic value as a result of their immunomodulatory effects. For example, the polypeptides of the present disclosure can be utilized in treating primary and secondary infections, modulating the deleterious effects of radiotherapy and chemotherapy, accelerating the rate of wound healing, decreasing some of the effects of aging, and improving the efficacy of other treatments. In addition, the polypeptides of the present disclosure may be utilized as an adjuvant in surgery and treatments using antifungal, antibiotic, and antiviral agents (e.g. utilized in the treatment of hepatitis B and hepatitis C).
It should be appreciated that the therapeutically effective dosage for the enhancement of immune functions in vivo may be extrapolated from in vitro assays (e.g. E-Rosette Assay) using the polypeptides and/or compositions of the present disclosure alone or in combination with other immunoregulating factors.
The preclinical and clinical therapeutic use of the subject matter of the present disclosure in the treatment of disease or the enhancement of immune functions will be best accomplished by those of skill, employing accepted principles of diagnosis and treatment. Accordingly, it should be appreciated that the compounds or polypeptides of the present disclosure can be utilized in a, for example, immunomodulating or immunoregulating composition. For example, the polypeptides of the present disclosure can be utilized in a immunopotentiating composition for reconstituting an immune function in a mammal. For example, a method for reconstituting an immune function in a mammal can comprise administering to the mammal an immunopotentiating effective amount of a compound or polypeptide having an amino acid sequence as follows: X-Ser-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 (SEQ ID NO:2) or a variant of the amino acid sequence that is (i) at least about 70% or more (e.g. 75%, 80%, 85%, 90%, or 95%) homologous to the amino acid sequence and (ii) has immunopotentiating activity in an E-Rosette forming assay, where (i) X is an acetyl group or H and (ii) at least one peptide bond between amino acids of the amino acid sequence or variant thereof is formed between a pair of functional groups selected from the group consisting of β-carboxy group of Asp and α-amino group of Ala, β-carboxy group of Asp and α-amino group of Thr, β-carboxy group of Asp and α-amino group of Leu, γ-carboxy group of Glu and α-amino group of He, α-carboxy group of Glu and ε-amino group of Lys, γ-carboxy group of Glu and α-amino group of Lys, γ-carboxy group of Glu and ε-amino group of Lys, γ-carboxy group of Glu and α-amino group of Val, γ-carboxy group of Glu and α-amino group of Glu, γ-carboxy group of Glu and α- amino group of Ala, γ-carboxy group of Glu and α-amino group of Asn, α-carboxy of Thr and ε-amino of Lys, α-carboxy of Leu and ε-amino of Lys, and α-carboxy of Lys and ε- amino of Lys.
As alluded to above, the present disclosure also provides pharmaceutical compositions. The pharmaceutical compositions of the present disclosure can be administered to any animal which may experience the beneficial effects of the compounds of the disclosure. Foremost among such animals are humans, although the disclosure is not intended to be so limited. The pharmaceutical compositions of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
The pharmaceutical compositions of the present disclosure may be administered by any method that achieves their intended purpose. For example, administration may be by parenteral , subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or buccal routes. Alternatively, or concurrently, administration may be by the oral route. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
In one embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of a Federal or state government, or listed in the U.S. Pharmacopeia or other generally recognized Pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerine, glycol or other synthetic solvents. Water can be utilized as a carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates. Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for the adjustment of tonicity such as sodium chloride or dextrose are also contemplated. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose made of glass or plastic.
These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides, microcrystalline cellulose, gum tragacanth or gelatin. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions will contain a therapeutically effective amount of a therapeutic (e.g. a polypeptide of the present disclosure), preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.
In one embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to an animal, such as a human being. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration. The amount of the polypeptide(s) of the disclosure which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro assays such as those discussed above may optionally be employed to help identify optimal dosage ranges. The dose ranges for the administration of the compositions of the present disclosure are those large enough to produce the desired effect, whereby, for example, an immunopotentiating effect is achieved. However, the doses should not be so large as to cause adverse side effects, such as unwanted cross reactions anaphalactic reactions and the like. Generally, as indicated above, the dosage will vary with the age, condition, sex and extent of the disease or condition in the patient . Counterindication, if any, immune tolerance and other variables will also affect the proper dosage. In addition, the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner in view of the particular circumstances. While the disclosure has been illustrated and described in detail in the foregoing description, such illustration and description is to be considered as exemplary and not restrictive in character, it being understood that only the preferred embodiments have been shown and described and that all changes and modifications that come within the spirit of the disclosure are desired to be protected.

Claims

CLAIMS:
1. A composition, comprising: a compound which includes amino acid sequence Ser-Asp-Ala-Ala-Val- Asp-Thr-Ser-Ser-Glu-Ile-Thr-Tl ι--Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu- Ala-Glu-Asn (SEQ ID NO:l) or a variant of the amino acid sequence that is at least about 70%) homologous to the amino acid sequence, wherein at least one peptide bond between amino acids of the amino acid sequence or variant thereof is formed between a pair of functional groups selected from the group consisting of β-carboxy group of Asp and α-amino group of Ala, β-carboxy group of Asp and α-amino group of Thr, β-carboxy group of Asp and α-amino group of Leu, γ-carboxy group of Glu and α-amino group of He, α-carboxy group of Glu and ε- amino group of Lys, γ-carboxy group of Glu and α-amino group of Lys, γ-carboxy group of Glu and ε-amino group of Lys, γ-carboxy group of Glu and α-amino group of Val, γ- carboxy group of Glu and α-amino group of Glu, γ-carboxy group of Glu and α-amino group of Ala, γ-carboxy group of Glu and α-amino group of Asn, α-carboxy of Thr and ε- amino of Lys, α-carboxy of Leu and ε-amino of Lys, α-carboxy of Lys and ε-amino of Lys, carboxy group of β-Ala and α-amino group of Ala, carboxy group of β-Ala and α- amino group of Glu, and carboxy group of β-Ala and α-amino group of Val.
2. The composition of claim 1, wherein: the at least one peptide bond between amino acids of the amino acid sequence or variant thereof is formed between β-carboxy group of Asp and α-amino group of Ala.
3. The composition of claim 1, wherein: the at least one peptide bond between amino acids of the amino acid sequence or variant thereof is formed between β-carboxy group of Asp and α-amino group of Thr.
4. The composition of claim 1, wherein: the at least one peptide bond between amino acids of the amino acid sequence or variant thereof is formed between β-carboxy group of Asp and α-amino group of Leu.
5. The composition of claim 1, wherein: the at least one peptide bond between amino acids of the amino acid sequence or variant thereof is formed between γ-carboxy group of Glu and α-amino group of He.
6. The composition of claim 1, wherein: the at least one peptide bond between amino acids of the amino acid sequence or variant thereof is formed between α-carboxy group of Glu and ε-amino group of Lys.
7. The composition of claim 1, wherein: the at least one peptide bond between amino acids of the amino acid sequence or variant thereof is formed between γ-carboxy group of Glu and α-amino group of Lys.
8. The composition of claim 1, wherein: the at least one peptide bond between amino acids of the amino acid sequence or variant thereof is formed between γ-carboxy group of Glu and ε-amino group of Lys.
9. The composition of claim 1, wherein: the at least one peptide bond between amino acids of the amino acid sequence or variant thereof is formed between γ-carboxy group of Glu and α-amino group of Val.
10. The composition of claim 1, wherein: the at least one peptide bond between amino acids of the amino acid sequence or variant thereof is formed between γ-carboxy group of Glu and α-amino group of Glu.
11. The composition of claim 1 , wherein: the at least one peptide bond between amino acids of the amino acid sequence or variant thereof is formed between γ-carboxy group of Glu and α-amino group of Ala.
12. The composition of claim 1, wherein: the at least one peptide bond between amino acids of the amino acid sequence or variant thereof is formed between γ-carboxy group of Glu and α-amino group of Asn.
13. The composition of claim 1, wherein: the at least one peptide bond between amino acids of the amino acid sequence or variant thereof is formed between α-carboxy of Thr and ε-amino of Lys.
14. The composition of claim 1, wherein: the at least one peptide bond between amino acids of the amino acid sequence or variant thereof is formed between α-carboxy of Leu and ε-amino of Lys.
15. The composition of claim 1, wherein: the at least one peptide bond between amino acids of the amino acid sequence or variant thereof is formed between α-carboxy of Lys and ε-amino of Lys.
16. The composition of claim 1, wherein: the at least one peptide bond between amino acids of the amino acid sequence or variant thereof is formed between carboxy group of β-Ala and α-amino group of Ala.
17. The composition of claim 1, wherein: the at least one peptide bond between amino acids of the amino acid sequence or variant thereof is formed between carboxy group of β-Ala and α-amino group of Glu.
18. The composition of claim 1, wherein: the at least one peptide bond between amino acids of the amino acid sequence or variant thereof is formed between and carboxy group of β-Ala and α-amino group of Val.
19. A composition, comprising: a compound having the amino acid sequence X-Ser-Asp-Ala-Ala-Val- Asp-Thr-Ser-Ser-Glu-Ile-Tlir-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu- Ala-Glu-Asn (SEQ ID NO:2) or a variant of the amino acid sequence that is at least about 70%) homologous to the amino acid sequence and has activity in an E-Rosette assay, wherein (i) X is an acetyl group or H and (ii) at least one peptide bond between amino acids of the amino acid sequence or variant thereof is formed between a pair of functional groups selected from the group consisting of β-carboxy group of Asp and α-amino group of Ala, β-carboxy group of Asp and α-amino group of Thr, β-carboxy group of Asp and α-amino group of Leu, γ-carboxy group of Glu and α-amino group of He, α-carboxy group of Glu and ε-amino group of Lys, γ-carboxy group of Glu and α- amino group of Lys, γ-carboxy group of Glu and ε-amino group of Lys, γ-carboxy group of Glu and α-amino group of Val, γ-carboxy group of Glu and α-amino group of Glu, γ- carboxy group of Glu and α-amino group of Ala, γ-carboxy group of Glu and α-amino group of Asn, α-carboxy of Thr and ε-amino of Lys, α-carboxy of Leu and ε-amino of Lys, α-carboxy of Lys and ε-amino of Lys, carboxy group of β-Ala and α-amino group of Ala, carboxy group of β-Ala and α-amino group of Glu, and carboxy group of β-Ala and α-amino group of Val.
20. The composition of claim 19, wherein: the pair of functional groups forming the at least one peptide bond between amino acids of the amino acid sequence or variant thereof is selected from the group consisting of β-carboxy group of Asp and α-amino group of Ala, β-carboxy group of Asp and α-amino group of Thr, and β-carboxy group of Asp and α-amino group of Leu.
21. The composition of claim 19, wherein: the pair of functional groups forming the at least one peptide bond between amino acids of the amino acid sequence or variant thereof is selected from the group consisting of γ-carboxy group of Glu and α-amino group of He, γ-carboxy group of Glu and α-amino group of Lys, γ-carboxy group of Glu and ε-amino group of Lys, γ- carboxy group of Glu and α-amino group of Val, γ-carboxy group of Glu and α-amino group of Glu, γ-carboxy group of Glu and α-amino group of Ala, and γ-carboxy group of Glu and α-amino group of Asn.
22. The composition of claim 19, wherein: the pair of functional groups forming the at least one peptide bond between amino acids of the amino acid sequence or variant thereof is selected from the group consisting of α-carboxy group of Glu and ε-amino group of Lys, α-carboxy of Thr and ε-amino of Lys, α-carboxy of Leu and ε-amino of Lys, and α-carboxy of Lys and ε- amino of Lys.
23. A polypeptide having an amino acid sequence as follows: Ser-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 (SEQ ID NO:l) or a variant of the amino acid sequence that is (i) at least about 70% homologous to the amino acid sequence and (ii) has activity in an E-Rosette assay, wherein at least one peptide bond between amino acids of the amino acid sequence or variant thereof is formed between a pair of functional groups selected from the group consisting of β-carboxy group of Asp and α-amino group of Ala, β-carboxy group of Asp and α-amino group of Thr, β-carboxy group of Asp and α-amino group of Leu, γ-carboxy group of Glu and α-amino group of He, α-carboxy group of Glu and ε- amino group of Lys, γ-carboxy group of Glu and α-amino group of Lys, γ-carboxy group of Glu and ε-amino group of Lys, γ-carboxy group of Glu and α-amino group of Val, γ- carboxy group of Glu and α-amino group of Glu, γ-carboxy group of Glu and α-amino group of Ala, γ-carboxy group of Glu and α-amino group of Asn, α-carboxy of Thr and ε- amino of Lys, α-carboxy of Leu and ε-amino of Lys, α-carboxy of Lys and ε-amino of Lys, carboxy group of β-Ala and α-amino group of Ala, carboxy group of β-Ala and α- amino group of Glu, and carboxy group of β-Ala and α-amino group of Val.
24. The polypeptide of claim 23, wherein: the at least one peptide bond between amino acids of the amino acid sequence or variant thereof is formed between β-carboxy group of Asp and α-amino group of Ala.
25. The polypeptide of claim 23 , wherein: the at least one peptide bond between amino acids of the amino acid sequence or variant thereof is formed between β-carboxy group of Asp and α-amino group of Thr.
26. The polypeptide of claim 23, wherein: the at least one peptide bond between amino acids of the amino acid sequence or variant thereof is formed between β-carboxy group of Asp and α-amino group of Leu.
27. The polypeptide of claim 23, wherein: the at least one peptide bond between amino acids of the amino acid sequence or variant thereof is formed between γ-carboxy group of Glu and α-amino group of He.
28. The polypeptide of claim 23, wherein: the at least one peptide bond between amino acids of the amino acid sequence or variant thereof is formed between α-carboxy group of Glu and ε-amino group of Lys.
29. The polypeptide of claim 23, wherein: the at least one peptide bond between amino acids of the amino acid sequence or variant thereof is formed between γ-carboxy group of Glu and α-amino group of Lys.
30. The polypeptide of claim 23, wherein: the at least one peptide bond between amino acids of the amino acid sequence or variant thereof is formed between γ-carboxy group of Glu and ε-amino group of Lys.
31. The polypeptide of claim 23 , wherein: the at least one peptide bond between amino acids of the amino acid sequence or variant thereof is formed between γ-carboxy group of Glu and α-amino group of Val.
32. The polypeptide of claim 23, wherein: the at least one peptide bond between amino acids of the amino acid sequence or variant thereof is formed between γ-carboxy group of Glu and α-amino group of Glu.
33. The polypeptide of claim 23, wherein: the at least one peptide bond between amino acids of the amino acid sequence or variant thereof is formed between γ-carboxy group of Glu and α-amino group of Ala.
34. The polypeptide of claim 23, wherein: the at least one peptide bond between amino acids of the amino acid sequence or variant thereof is formed between γ-carboxy group of Glu and α-amino group of Asn.
35. The polypeptide of claim 23, wherein: the at least one peptide bond between amino acids of the amino acid sequence or variant thereof is formed between α-carboxy of Thr and ε-amino of Lys.
36. The polypeptide of claim 23, wherein: the at least one peptide bond between amino acids of the amino acid sequence or variant thereof is formed between α-carboxy of Leu and ε-amino of Lys.
37. The polypeptide of claim 23, wherein: the at least one peptide bond between amino acids of the amino acid sequence or variant thereof is formed between α-carboxy of Lys and ε-amino of Lys.
38. The polypeptide of claim 23, wherein: the at least one peptide bond between amino acids of the amino acid sequence or variant thereof is formed between carboxy group of β-Ala and α-amino group of Ala.
39. The polypeptide of claim 23, wherein: the at least one peptide bond between amino acids of the amino acid sequence or variant thereof is formed between carboxy group of β-Ala and α-amino group of Glu.
40. The polypeptide of claim 23, wherein: the at least one peptide bond between amino acids of the amino acid sequence or variant thereof is formed between and carboxy group of β-Ala and α-amino group of Val.
41. A polypeptide comprising an amino acid sequence as follows: Ac-Ser- 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 (SEQ ID NO:3), wherein a peptide bond is formed between the β-carboxy group of Asp and the α-amino group of Ala, a peptide bond is formed between the β-carboxy group of Asp and the α-amino group of Thr, and a peptide bond is formed between the β-carboxy group of Asp and α-amino group of Leu.
42. A polypeptide comprising an amino acid sequence as follows:
Ac-Ser-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 (SEQ ID NO:3), wherein a peptide bond is formed between the β-carboxy group of Asp and the α-amino group of Ala.
43. A polypeptide comprising an amino acid sequence as follows: Ac-Ser-Asp-Ala-Ala-Val-Asp-Trrr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-
Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn (SEQ ID NO:3), wherein a peptide bond is formed between the β-carboxy group of Asp and the α-amino group of Thr.
44. A polypeptide comprising an amino acid sequence as follows: Ac-Ser-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 (SEQ ID NO:3), wherein a peptide bond is formed between the β-carboxy group of Asp and α-amino group of Leu.
45. A polypeptide comprising an amino acid sequence as follows: Ac-Ser-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 (SEQ ID NO:3), wherein a peptide bond is formed between the β-carboxy group of Asp and the α-amino group of Ala and a peptide bond is formed between the β-carboxy group of Asp and the α-amino group of Thr.
46. A polypeptide comprising an amino acid sequence as follows:
Ac-Ser-Asp-Ala-Ala-Val-Asρ-Thr-Ser-Ser-Glu-Ile-Tfrr-Thr-Lys-Asp-Leu- Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn (SEQ ID NO:3), wherein a peptide bond is formed between the β-carboxy group of Asp and the α-amino group of Ala and a peptide bond is formed between the β-carboxy group of Asp and α-amino group of Leu.
47. A polypeptide comprising an amino acid sequence as follows: Ac-Ser- 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 (SEQ ID NO:3), wherein a peptide bond is formed between the β-carboxy group of Asp and the α-amino group of Thr and a peptide bond is formed between the β-carboxy group of Asp and α-amino group of Leu.
48. A method for reconstituting an immune function in a mammal, the method comprising: administering to the mammal an immunopotentiating effective amount of a compound having an amino acid sequence as follows:
X-Ser-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 (SEQ ID NO:2) or a variant of the amino acid sequence that is (i) at least about 70%) homologous to the amino acid sequence and (ii) has activity in an E-Rosette assay, wherein (i) X is an acetyl group or H and (ii) at least one peptide bond between amino acids of the amino acid sequence or variant thereof is formed between a pair of functional groups selected from the group consisting of β-carboxy group of Asp and α-amino group of Ala, β-carboxy group of Asp and α-amino group of Thr, β-carboxy group of Asp and α-amino group of Leu, γ-carboxy group of Glu and α-amino group of Ile, α-carboxy group of Glu and ε-amino group of Lys, γ-carboxy group of Glu and α- amino group of Lys, γ-carboxy group of Glu and ε-amino group of Lys, γ-carboxy group of Glu and α-amino group of Val, γ-carboxy group of Glu and α-amino group of Glu, γ- carboxy group of Glu and α-amino group of Ala, γ-carboxy group of Glu and α-amino group of Asn, α-carboxy of Thr and ε-amino of Lys, α-carboxy of Leu and ε-amino of Lys, α-carboxy of Lys and ε-amino of Lys, carboxy group of β-Ala and α-amino group of Ala, carboxy group of β-Ala and α-amino group of Glu, and carboxy group of β-Ala and α-amino group of Val.
49. A compound having the formula:
Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Trrr-Thr-Lys-Asp-Leu- Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn (SEQ ID NO:3), wherein a peptide bond is formed between the β-carboxy group of Asp and the α-amino group of Ala, a peptide bond is formed between the β-carboxy group of Asp and the α-amino group of Thr, and a peptide bond is formed between the β-carboxy group of Asp and α-amino group of Leu.
50. A compound having the formula: Ac-Ser-Asp-Ala-Ala-Val-Asp-Thι--Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu- Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn (SEQ ID NO:3), wherein a peptide bond is formed between the β-carboxy group of Asp and the α-amino group of Ala.
51. A compound having the formula: Ac-Ser-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 (SEQ ID NO:3), wherein a peptide bond is formed between the β-carboxy group of Asp and the α-amino group of Thr.
52. A compound having the formula: Ac-Ser-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 (SEQ ID NO:3), wherein a peptide bond is formed between the β-carboxy group of Asp and α-amino group of Leu.
53. A compound having the formula: Ac-Ser-Asp-Ala-Ala-Val-Asp-Tlir-Ser-Ser-Glu-Ile-Tlπ-Thr-Lys-Asp-Leu-
Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn (SEQ ID NO:3), wherein a peptide bond is formed between the β-carboxy group of Asp and the α-amino group of Ala and a peptide bond is formed between the β-carboxy group of Asp and the α-amino group of Thr.
54. A compound having the formula: Ac-Ser-Asp-Ala-Ala-Val-Asp-Tlir-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-
Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn (SEQ ID NO:3), wherein a peptide bond is formed between the β-carboxy group of Asp and the α-amino group of Ala and a peptide bond is formed between the β-carboxy group of Asp and α-amino group of Leu.
55. A compound having the formula: Ac-Ser-Asp-Ala-Ala-Val-Asp-Tl r-Ser-Ser-Glu-He-Thι--Thr-Lys-Asp-Leu-
Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu- Ala-Glu-Asn (SEQ ID NO: 3), wherein a peptide bond is formed between the β-carboxy group of Asp and the α-amino group of Thr and a peptide bond is formed between the β-carboxy group of Asp and α-amino group of Leu.
PCT/US2003/002627 2003-01-29 2003-01-29 Immunoregulating compounds and an associated method WO2004069270A1 (en)

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WO2000069900A2 (en) * 1999-05-17 2000-11-23 Conjuchem, Inc. Protection of endogenous therapeutic peptides from peptidase activity through conjugation to blood components

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WO2000069900A2 (en) * 1999-05-17 2000-11-23 Conjuchem, Inc. Protection of endogenous therapeutic peptides from peptidase activity through conjugation to blood components

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