Classifications

• • 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
• • 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
  • C07K14/66—Thymopoietins
  • C07K14/662—Thymopoietins at least 1 amino acid in D-form
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/02—Linear peptides containing at least one abnormal peptide link
• • 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 invention relates, generally, to synthetic immunoactive peptides having immunodulating and therapeutic activities and use of such peptides in pharmaceuticals. More particularly, the present invention relates to chemically synthesized immuno- active peptides useful in immunotherapy for regulation of T-cell dependent immunity.
• Fraction V the uncharacterized mixture of poly ⁇ peptides from calf thymus extract, having relatively high amounts of alpha-, beta- and gamma-thymosins and related polypeptides, has proven to be useful from immunomodulation in in vitro and in vivo tests, as summarized in Aiuti, F. and Wigzell, H. , Thymus. Thymic Hormones, and T-Lymphocytes, Proceedings of the Serono Symposium, V. 38 (Academic Press 1989.)
• the potential effectiveness of Fraction V as an immunomodulating drug has been disclosed in H. Strausser, U.S. Patent No. 4,444,757, issued April 24th, 1984.
• the clinical use of the foregoing preparations has been limited by:
• Pentapeptide sequences with thymopoietin-like activity and increased resistance to enzymatic degradation in biological fluids were created and disclosed in G. Goldstein et al. , U.S. Patent No. 4,505,853, issued March 19th, 1985, however, their biological activity was comparable only with activity of parental peptide sequences.
• an object of the present invention to provide a series of synthetic peptides which are effective in the therapy of immunodeficien ⁇ cies, immunosuppression and T-cell subset deviations, and related ailments.
• X is a hydrophobic or neutral amino acid independently selected from the group consisting of Ala, D-Ala, Gly, D-Gly, Ile, 5-Ile, Leu, D-Leu, Met, D-Met, Phe, D-Phe, Pro, #-Pro, Sar, D-Sar, Ser, D-Ser, Tre, D-Tre, Trp,
• Ai through A12 are, independently, neutral or positively-side-chain-charged amino acids independently selected from the group consisting of Ala, D-Ala, Arg, D-Ar , Asp, D-Asp, Glu, D-Glu , Gly, D-Gly, His, J-His, Ile, D-Ile, Leu, D-Leu, Met, D-Met, Phe, D-Phe, Pro, D-Pro, dehydro Pro, Sar, D-Sar, Ser, D-Ser, Tre, D-Tre, Trp, D-Trp, Val and D-Val;
• Bi through B12 are, independently, neutral or negatively-side-chain-charged amino acids independently selected from the group consisting of Ala, D-Ala , Asp, D-Asp, Glu, D-Glu, Gly, D-Gly, Ile, D-Ile, Leu, D-Leu, Met, D-Met.
• the foregoing peptides in accordance with the present invention, may be acylated and/or amidated.
• the foregoing generic formula includes the following sequences of amino acids in accordance with the foregoing definitions for "A,” “B” and "X";
• A1A2B2XA3B3 2. A1A2XB2A2A3 3. A1B1XB2A2A3 4. B1A1XB2A2A3
• A1B2A2XA3B3 10. A1B2A2XB3A3
• A1A2A3B3XB4A4 18. Ai A2 A3 B3 XA4 B4
• BiAiA2B2XB3A3B A4 58.
• Ai BiA2 B2XB3A3A4B4 59.
• BiAiA2B2XA3B3B4A4 60.
• BiAiXA2 B2B3A3XB4A4 86.
• BiAiXB2A2 B3A3XA4 B4 The present invention should be understood as encompassing pharmaceutically acceptable acid or base salts, as well as the free peptides generically described above and which are detailed hereinafter.
• amino acid sequence No. 41 provides the most effective peptides.
• Amino acid sequences Nos. 49 and 59 have also been found to be very effective.
• compositions to be administered to a patient in the treatment of immunodeficiencies, immunosuppression, T-cell subset deviations and for the enhancement of vaccinations, etc. is to include one or more of the foregoing peptides in combination with an appropriate solubilizer.
• Suitable additives known to those skilled in the art e.g., carriers, preservatives and viscosity modifiers, may be added to the compounds of the invention as required.
• the synthetic immunologically-active peptides of the present invention are, for example, for parenteral use, direct intranasal, ear, eye, intra- vaginal or rectal instillation and application to intact or injured conjunctive, mucosa or skin, in order to accomplish the normalization of immune responses.
• the preparations are to be used either locally or systemically in order to induce immuno- modulation, enhance the effect of vaccination and achieve other goals of immunotherapy.
• Preferred peptides of the present invention are those wherein:
• Ai through An are, independently, Arg, Asn, Gin, Lys, Phe or Val;
• Bi through Bn are, independently, Asp, Glu, Tyr, Phe or Val ;
• X is Ala, Gly, Ile, Leu, Phe or Val.
• Most preferred peptides coming within the scope of the present invention contain either balanced side chain charges of the sequences which are symmetical relative to X or uncompensated opposite side chain charges relative to X.
• peptides may be acylated, amidated or cyclized during synthesis.
• peptides synthesized in accordance with the present invention which have been found to be most effective in immunotherapy, include:
• peptides synthesized in accordance with the present invention which have been found to be most effective in immunotherapy, include:
• Cyclized peptides synthesized in accordance with the present invention include:
• peptides encompassed within the scope of the present invention and which have effective levels of immunotherapeutic activity, though not as great as those peptides listed above, further include:
• Linear peptides of the present invention may be synthesized via procedures generally known to those skilled in the art and which are of wide use at present. M. Bodanszky and A. Bodanszky, Peptide Chemistry: A Practical Textbook. Springer-Verla , N.Y. (1988); M. Bodanszky and A. Bodanszky, The Practice of Peptide Synthesis. Springer-Verla . N.Y. (1989). All linear peptides of the present invention, for example, may be synthesized by the solid phase method utilizing peptide synthesizers which are commercially available through, for example, Applied Biosystems, Inc., Foster City, California, U.S.A., and t-Boc chemistry.
• Cyclized peptides synthesized in accordance with the present invention may be synthesized in accordance with the following:
• Dicarbonic amino acids were attached to a resin via estification of a side chain carboxyl group and deprotected, Aminoacid-O-nitrophenol esters and aminoacid-O-succinimide esters were coupled directly to the deprotected alpha-amino groups of the growth peptide in the desired sequence to produce C-terminal and N-terminal deprotected peptide-resin.
• These resin-bounded peptides were cyclized using dicyclo- hexyl carbodiimide, cleaved and side chain deprotected following purification. Aminoacidic analysis or NMR spectra may be utilized to confirm that the desired cyclized peptide has been produced.
• Figures 1 - 5 (i.e., Tables 1 - 5) present experimental data illustrating the use and effective ⁇ ness of the peptides of the present invention.
• Phytohemagglutinin is a non ⁇ specific stimulator of T-lymphocyte activation, which does not induce 3 H-thymidine uptake by peripheral blood lymphocytes in sub-optimal amounts.
• Peptides of the present invention were added in a wide range of concentrations to lymphocyte cultures and caused a dramatic increase of 3 H-thymidine incorporation into cell DNA.
• the peptides of the invention are over 100 to 1000 times more active than other known immunologically-active sequences.
• the peptides tested in Table 1 were as follows:
• Peripheral blood lymphocytes were purified by isopicnic centrifugation, washed and diluted in complete RPMI 1640 medium, supplemented with human AB sera. IO 5 cells were added into the wells of 96 well plates, which contain 100 ⁇ l of tested substances, diluted in RPMI 1640 culture medium as above, and a suboptimal amount of PHA. Cells were cultured for 4 days and 3 H-thymidine was added into each well for 24 hours. Cells were harvested and incorporation of 3 H-thymidine was measured by scintillation counting. All experiments were conducted in quadruplicate.
• vaccinia virus partially protects the mouse from infection with Ectromelia virus.
• the latter infection is lethal in most cases in inbred mice.
• Mice BALB/c received peptides of the present invention in doses of from 0.1 meg to 1 meg per kg of body weight, one day prior to exposure to vaccinia virus. A month later, they were injected with Ectromelia virus in an extract from the spleen and liver of spontaneously infected animals.
• the clinical signs of infection on record were hepatosplenomegaly, mucosal inflammation and necrosis of extremities and tail. The potentiating effect of peptides following vaccination with viccinia virus was evident.
• mice C57BL/6 with Influenza A virus strain WSN saved a significant number of animals. (See, Table 5.)
• the peptide had the amino acid sequence of Tyr-Arg-Lys-Glu-Leu-Glu-Lys-Lys-Glu. Mortality was recorded during a two-week period. P was calculated using the "F" distribution of Fischer.
• Peptides of the present invention injected in doses of from 0.1 meg to 1 meg per kg of mouse body weight, normalized the depleted T-cell immunity of thymectomized animals.
• the medical formulations of the present invention preferably include an effective amount of one or more peptides and a solubilizer, with the possible inclusion of additional carriers and preservatives, as determined by the specifics of the different product formulations so desired by the skilled artisan.
• Solubilizers useful in combination with the peptides of the present invention include any solubilizer which is compatible with bodily fluids.
• solubilizers are water, solvents such as dimethylsulfoxide, propylene glycol, dimethylform- a ide and mixtures thereof, and surface active agents, such as non-ionic alkylene oxide block copolymers.
• additives can be used to achieve physiological concentrations of inorganic salts, normal osmotic pressure and effective lyophilization.
• Such additives can be selected from, for example, sodium chloride and potassium chloride, sodium and potassium phosphate salts, sucrose, glucose, protein hydro- lysate, dextran, polyvinylpyrrolidone and poly ⁇ ethylene glycol, among others.
• peptides are included in medical treatment composition of the present invention in- order to provide doses ranging from 0.0001 mg to about 5 mg per kg of body weight for parenteral use, as well as concentrations ranging from about 0.0001% to about 5% for topical use.
• a preferred composition of the present invention for parenteral usage is a 0.01% solution of peptides in 1 ml of 0.85 wt-% sodium chloride containing 0.1 wt-% of ascorbic acid; it can be lyophilized in the presence of 100 mg glucose.
• a preferred composition of the present invention for eye and ear drops is a composition comprising a 0.0001 wt-% solution of peptides in a 0.85% sodium chloride solution.
• compositions utilizing the peptides of the present invention may be used for immunomodulation of various immunodeficiencies and immunosuppressed conditions, T-cell subset and lymphocyte deviations, enhancement of a vaccine's efficacy, as well as for immunotherapy, including infections, local or systemic complications of non- infectious diseases, postoperatives inflammations, wounds and burns. While only several embodiments of the present invention have been described, it will be obvious to those of ordinary skill in the art that many modifications may be made to the present invention without departing from the spirit and scope thereof.

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• 1991
  • 1991-11-22 WO PCT/US1991/008795 patent/WO1992009628A1/en not_active Application Discontinuation
  • 1991-11-22 AU AU91408/91A patent/AU9140891A/en not_active Abandoned
  • 1991-11-22 EP EP19920902085 patent/EP0563212A1/de not_active Withdrawn

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