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
• • 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
  • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
  • C07K14/43509—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from crustaceans
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/04—Antineoplastic agents specific for metastasis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• • 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 falls within the field of cancer immunotherapy.
• peptides or combinations thereof from region 32-51 of the Limulus anti-LPS Factor protein, which do not bind lipopolysaccharides and have anti-tumor and immunomodulatory properties useful for the treatment of cancer and metastasis.
• TLR7 agonist Immune system activation by a TLR7 agonist is in a phase I clinical trial with promising results as a new drug for the treatment of melanoma and other tumors (Dudek AZ, et al (2005) ASCO Annual Meeting).
• the aforementioned agonists of TLR9 and TLR7 are also being evaluated in viral infections, given their ability to promote an effective immune response in the host.
• the so-called Heat shock protein (Hsp) which bind to TLR4, have been developed and marketed as a fusion product with the E7 oncoprotein.
• This new immunotherapeutic approach is also known as therapeutic vaccines and has a broad perspective on the therapy of diseases related to the human papillomavirus (Chu NR et al., (2000) Immunotherapy of a human papillomavirus (HPV) type 16 E7-expressing tumor by administration of fusion protein comprising Mycobacterium bovis bacille Calmette-Guerin (BCG) hsp65 and HPV16 E7. Clin Exp Immunol 121: 216-225).
• ToII type receptors are receptors that are found in cells of the immune system and recognize molecular patterns associated with pathogens example: lipopolysaccharides (in English Lipopolysaccharide, abbreviated LPS), lipotecoic acid, non-methylated CpG sequences, double and single stranded viral RNA.
• LPS lipopolysaccharides
• CpG sequences non-methylated CpG sequences
• double and single stranded viral RNA double and single stranded viral RNA.
• the recognition of the invading pathogen by the TLRs helps the immune system to direct a type of Th1 / Th2 shared response that allows the organism to efficiently eradicate the infection.
• the use of TLR agonists as cancer drugs is based on the activation of the innate and adaptive immune system.
• Dendritic cells Dendritic CeIIs are professional antigen presenting cells, whose main role is to link the innate and adaptive immune response through direct cell-cell interaction and the production of cytokines.
• the DC can be of myeloid or lymphoid origin attending to the expression of a series of surface markers, as well as to the expression of different TLRs.
• DCs of lymphoid origin known as dendr cells Plasmacytic ticas are the main producers of type I IFNs.
• dendritic cells have been manipulated as a promising cell adjuvant for the development of therapeutic vaccines against cancer and chronic viral infections (Santini SM, et al (2003 ) A new type I IFN- mediated pathway for rapid differentiattion of monocytes into highly active dendritic cells. Stem CeIIs, 21: 357-362).
• This is a highly expensive and difficult technique, which is why we are currently working on finding strategies more practical and less expensive alternatives (Van Epps HL (2005) New hope for tumor vaccines. The Journal of Experimental Medicine, Vol. 202: 1615).
• Interferons of type I have recently shown to exert important effects on the immune system, including the promotion of cellular and humoral responses mediated by its adjuvant effect on dendritic cells (Bogdan, C . (2000) The function of type I interferons in antimicrobial immunity. Curr, Opin Immunol. 12: 419-424). Recent works show a critical function for endogenously produced type I interferons, in the regression processes of a highly immunogenic syngeneic murine sarcoma and in protecting the host from the formation of carcinogenic primary tumors (Gavin P. Dunn, et al. (2005 ) A criticism! Function for type I interferons in cancer immunoediting.
• Interferon alpha plays an important role in the initiation of the viral response of T lymphocytes, via a direct activation of CD4 + and CD8 + T cells in viral infections such as Influenza (Fonteneau J. F, et al. (2003 ) Activation of influenza virus-specific CD + 4 and CD + 8 T cells: a new role for plasmacytoid dendritic cells in adaptive immunity.
• Hoess WO 95/05393
• WO 95/05393 relates his invention to substances which bind with great affinity to the LPS and are useful for the prevention or treatment of for example: sepsis mediated by Gram-negative and Gram-positive bacteria and for the treatment of bacterial infections in general and by fungi.
• These substances are peptides that bind LPS containing a binding domain for the endotoxin (Hoess A., et al, (1993) Crystal structure of an endotoxin-neutralizing protein from the horseshoe crab, Limulus anti-LPS factor, at 1.5A ° resolution The EMBO J. 12: 3351-3356).
• Vallesp ⁇ (US 6,191, 114) relates his invention to the peptide comprising amino acids 31-52 of the LALF protein, which exerts an antiviral effect on Hep-2 and MDBK cells by virtue of the induction of IFN- ⁇ and ⁇ ; as well as relates his invention to the use of said peptide for the treatment of viral infections and disorders related to immunosuppression.
• the same author has demonstrated the anti-infective effect of this peptide in animal models of sepsis, (Vallespi MG, et all (2003) A Limulus anti-LPS factor-derived peptide modulates cytokine gene expression and promotes resolution of bacterial acute infection in mice International Immunopharmacology, 3: 247-256).
• the present invention solves the aforementioned problem, by providing peptides from the sequence HYRIKPTFRRLKWKKYKGKFW (SEQ. ID. NO: 13) corresponding to region 32-51 of the LALF protein, in which amino acid substitutions have been introduced which guarantee eliminating the ability to bind to the LPS and enhance the anti-tumor and immunomodulatory effect.
• Analog peptides derived from said substitutions which do not bind LPS or heparin and show a greater anti-tumor and immunomodulatory effect than the parental peptide have the following sequences:
• HARIKPTFRRLKWKYKGKFW (SEQ. ID. NO: 1)
• HYRIKPTARRLKWKYKGKFW (SEQ. ID. NO: 2)
• HYRIKPTFRRLAWKYKGKFW SEQ. ID. NO: 3
• HYRIKPTFRRLKWKYKGKFA SEQ. ID. NO: 4
• the LPS binding capacity of the peptides described by Hoess and Vallesp ⁇ have a disadvantage that their administration in the presence of LPS diverts the cytokine pattern from a shared Th1 / Th2 profile to a predominantly Th2, which would be contraindicated in cancer patients. These patients generally present concomitant infections due to their immunosuppressed state, for which reason the presence of LPS particles in blood is not ruled out.
• the administration of a peptide that can induce a predominant Th2 response type in the presence of the endotoxin would be an undesirable effect that would further deteriorate the patient's immune status, worsening the host response against the tumor.
• the invention also includes peptides, where two or more amino acid residues are substituted by alanine and comprise the amino acid sequences: HYRIKPTARRLAWKYKGKFW (SEQ. ID. NO: 8)
• HARIKPTARRLKWKYKGKFW SEQ. ID. NO: 9
• HARIKPTFRRLAWKYKGKFW SEQ. ID. NO: 10
• HARIKPTARRLAWKYKGKFW SEQ. ID. NO: 11
• HARIKPTARRLAWKYKGKFA SEQ. ID no. , which has been obtained by synthetic or recombinant route, as well as any fusion peptide that contains them.
• Homologous variant means any peptide that does not have the ability to bind LPS or heparin and maintains an anti-tumor and immunomodulatory effect.
• a mimetic variant means any molecule of chemical origin (non-protein) whose structure does not have the ability to bind LPS or heparin and maintains an anti-tumor and immunomodulatory effect.
• the pharmaceutical composition contains one or more of the peptides and chemical compounds or their pharmaceutically acceptable salts, as well as pharmaceutically acceptable excipients or vehicles.
• the pharmaceutical composition additionally contains an antigen selected from the group consisting of a bacterial, viral and cancer antigen.
• the peptides of the invention can be used in combination with conventional cancer treatments, such as chemotherapy, surgery, radiation, etc.
• Also part of the present invention is the use of peptides and chemical compounds for the preparation of a pharmaceutical composition for the treatment and / or the prevention of immunological disorders, in which it is necessary to develop an effective Th1 type immune response; treatment and / or prevention of cancer, as well as to develop an effective immune response to infections of bacterial and viral origin.
• the described peptides were defined by their ability not to bind the LPS, since the original HYRIKPTFRRLKWKYKGKFW sequence is previously described as optimal consensus domain for LPS binding (Hoess et al., (1993) Crystal structure of an endotoxin-neutralizing protein from the horseshoe crab, Limulus anti-LPS factor, at 1.5A ° resolution. The EMBO J. 12: 3351- 3356).
• the peptides described in this invention enhance the immunomodulatory effect given by the expression of IFN- ⁇ , compared to the LALF protein peptide comprising amino acids 31-52, which Vallesp ⁇ in his invention (US 6,191, 114) referred to as an anti-viral and immunomodulatory peptide.
• the peptides described in this invention can be administered to patients who are immunosuppressed and need an activation of the immune system's responsiveness eg; patients with acquired immunodeficiency syndrome (AIDS), patients undergoing complex surgeries.
• AIDS acquired immunodeficiency syndrome
• experimental data in vivo demonstrate the effectiveness of analogous peptides in tumors already implanted in mice, from murine B16 melanoma cells; Malignant lung epithelial cells derived from C57BL / 6 mice and 3LL-D122 cells from mouse lung cancer.
• the administration of the peptides can be used to reduce metastatic events.
• the described peptides can be used alone or in combination with current therapies for the treatment of cancer, as an example: surgery, radiation, chemotherapy.
• the peptides described in this invention administered prophylactically leads to a rapid innate immune response against the tumor, with a subsequent development of a specific antigenic adaptive immune response, emphasizing its use in prophylactic or therapeutic vaccines against cancer.
• Figure 1 Effect of the peptides on the ability to bind bacterial lipopolysaccharide (LPS).
• Figure 1A these experiments were performed in triplicate, the inhibition curves of an experiment are shown. The percent inhibition shown in Figure 1B is the average of three independent experiments.
• Figure 2 Effect of the peptides on the ability to bind the anionic compound heparin. The average of three independent experiments is shown.
• Figure 3 Effect of the peptides on the production of IFN ⁇ . ⁇ and IL-12 in human mononuclear cells. Three experiments were conducted with different donors. The results of a donor are shown.
• Figure 4 Antitumor effect of peptides in the TC-1 tumor model.
• Figure 5 Antitumor effect of peptide L-2 in the melanoma model.
• Figure 6 Antitumor effect of the L-2 analog peptide in prophylactic administration scheme in the TC-L tumor model
• Figure 7 Antitumor effect of the L-2 analog peptide in a double challenge scheme with the TC-1 tumor cells.
• Figure 8 Anti-metastatic capacity of the L-2 analog peptide.
• Figure 9 Effect of the L-2 analog peptide on the proliferation of TC-1, H-125 and L929 cells. DETAILED EXHIBITION OF REALIZATION MODES / EXAMPLES
• the peptides of the invention are synthesized using a solid phase procedure.
• the crude peptide is extracted with a 30% acetic acid solution, lyopholized and subsequently purified by RP-HPLC.
• the molecular mass of the purified peptides are verified using a JEOL JMS-HX110HF mass spectrometer with a FAB gun.
• the resulting preparation is non-antigenic, non-pyrogenic and pharmaceutically acceptable for administration in animals and humans.
• the substitutions were made promptly by introducing the amino acid alanine in each of the positions of the original HYRIKPTFRRLKWKYKGKFW sequence.
• Example 2 Selection of analog peptides that do not have lipopolysaccharide binding capacity (LPS).
• LPS lipopolysaccharide binding capacity
• the binding of analogous peptides to the LPS fixed to the solid surface was evaluated by direct competition with LALF peptide 32 -5 1 labeled with biotin (LALF 32-5 i-biotin) at a concentration of 0.2 uM, for Ia which was obtained 90% of the maximum union to the LPS.
• LALF 32-5 i-biotin biotin
• different concentrations of the analogous peptides were used from 10 uM to 0.01 uM as control the experiment was performed the curve of the LALF 32-51.
• the LALF3 2- 5i-b ⁇ ot ⁇ na in the presence of the analogous peptides incubated for 2 h at 37 0 C and then the plates were washed 5 times with washing solution.
• LALF 32-5 i-biotin bound to the LPS was detected by incubation for 45 min at 37 0 C with peroxidase - conjugated streptavidin (streptavidin-peroxidase) diluted 1: 2000.
• the plates were washed 5 times with wash solution and then the substrate solution (0.05 M citrate phosphate buffer pH 5.5, 1 tablet of 3,3,5,5-tetramethylbenzidine, 0.025% hydrogen peroxide) was added. It was incubated for an additional 15 minutes and the reaction was stopped by the addition of 2M sulfuric acid.
• the absorbance at 450 nm was quantified using a plate reader (Sensident Sean). There is a correlation between the optical density values and the LPS binding capacity of the analogous peptides. Analog peptides with greater capacity to bind to the LPS have inhibition curves with lower OD values with respect to the LALF 32- Si inhibition curve.
• Analog peptides with a lower capacity of binding to the LPS have inhibition curves with higher OD values with respect to the LALF 32- Si inhibition curve.
• the results of this example set forth in Figure 1A showed that peptides called L-2, L-8, L-12 and L-20 lose the ability to bind to LPS, however peptides L-9 and L-19 they demonstrate a capacity of union to the LPS similar to the LALF 32-S i.
• the analog L-3 shows a greater capacity of binding to the LPS.
• Figure 1B shows the percent inhibition of the analogous peptides, at a fixed concentration of 0.5 ⁇ M, in terms of the ability to displace the binding of LALF 32-5 i-biotin (0.2 ⁇ M) to the surface-fixed LPS solid.
• Example 3 Evaluation of the heparin binding of analog peptides L-2, L-8, L-12 and L-20.
• This test consists of an ELISA type competence system, similar to the one described above. 32- LALF peptide 5i-biotin in PBS 1X fixed polystyrene plates (Costar, USA) and all Ia was incubated overnight at 4 0 C. The peptides analogues L-
• L-8, L-12 and L-20 at a molarity of 2 ⁇ M were mixed with 250 units of heparin (sodium heparin, 5000 U / mL, Liorad) in PBS + 0.1% bovine albumin
• Example 4 Effect of analog peptides L-2, L-8, L-12 and L-20 on the expression of IFN- ⁇ , IFN- ⁇ and I L-12 in human mononuclear cells.
• RNA extraction was performed by the TriReagent method.
• the expression of the genes for IFN- ⁇ , IFN- ⁇ and I L-12 was determined by the technique of reverse transcription reaction and amplification by PCR (Perkin Elmer RT-PCR Kit). The results are shown as relative levels of messenger RNA normalized with the ⁇ -actin gene.
• the results obtained in this test demonstrated that the peptides L-2, L-8, L-12 and L-20 described in this invention are capable of inducing the expression of the genes for IFN- ⁇ and I L-12, Figure 3A .
• the analog peptides L-2, L-8 and L-12 are particularly more effective in inducing the gene for IFN- ⁇ than the LALF 32-S i peptide, Figure 3B.
• This example demonstrates that amino acid substitutions in the original 32-51 sequence of the LALF protein eliminates the ability to bind to the LPS and enhances the immunomodulatory effect.
• Example 5 Antitumor effect of analog peptides L-2, L-8, L-12 and L-20 in the tumor model TC-1.
• TC-1 cells derived from malignant C57BL / 6 lung epithelial cells were used, which were resuspended in saline solution (PBS).
• An amount of 50,000 cells in a volume of 200 ⁇ L was inoculated to the mice subcutaneously in the right hind leg.
• the 1 st peptide administration was performed subcutaneously in the right flank once tumors reached 100 mm 3 volume, Ia 2 nd injection was performed 10 days later.
• Example 6 Antitumor effect of the L-2 analog peptide in the melanoma model.
• MB16-F10 cells were used which were resuspended in saline solution (PBS).
• An amount of 15,000 cells in a volume of 200 ⁇ L was inoculated to the mice subcutaneously in the right hind leg.
• the peptide L-2 was administered, the second injection was made 7 days after the 1st injection and 14 days later the third injection was made.
• a dose of 4 mg of peptide was evaluated per kg of weight.
• Example 7 Antitumor effect of the L-2 analog peptide in prophylactic treatment scheme in the TC-1 tumor model.
• the TC-1 cells were used, which were resuspended in saline solution (PBS).
• PBS saline solution
• the mice received a first injection of the L-2 peptide (4 mg of peptide per kg of weight), after 7 days they received a second injection using the same dose; After 14 days of the first peptide injection, the animals were inoculated with 50 000 TC-1 cells in a volume of 200 ⁇ L of PBS subcutaneously in the right hind leg.
• the parameters evaluated to measure the antitumor effect of the peptide of interest were the time of tumor loosening, figure 6A and the survival of the animals, figure 6B.
• the results obtained in this test demonstrated that the L-2 peptide of this invention is effective in preventing the development of the tumor and increasing the survival of the animals. These results show that the peptide of this invention has a prophylactic effect, preventing the appearance of the tumor.
• Example 9 Antitumor effect of the L-2 analog peptide, in the Lewis Carcinoma metastasis model.
• Example 10 Effect of the L-2 analog peptide on tumor cell growth.
• TC-1, H-125 (non-small human lung cancer cells) and L929 (murine fibroblast) cells were seeded in 96-well plates (Costar) at a density of 2 x 10 4 cells / ml in Dulbecco medium (DMEM) (Gibco) supplemented with Fetal Bovine Serum (Gibco).
• DMEM Dulbecco medium
• Fetal Bovine Serum Gibco
• the peptides were added to the culture medium in a dose range between 9 ⁇ M and 300 ⁇ M. Incubation was performed for 72 hours in the presence of CO 2 at 5% and at the end of it was revealed with crystal violet. The plates were washed extensively with running water and finally the reading of the plate was carried out at an absorbance of 562 nm.

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Organic Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Medicinal Chemistry (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Gastroenterology & Hepatology (AREA)
• Immunology (AREA)
• Biophysics (AREA)
• Biochemistry (AREA)
• Genetics & Genomics (AREA)
• Zoology (AREA)
• Molecular Biology (AREA)
• Veterinary Medicine (AREA)
• Public Health (AREA)
• Animal Behavior & Ethology (AREA)
• Pharmacology & Pharmacy (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Toxicology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Engineering & Computer Science (AREA)
• Tropical Medicine & Parasitology (AREA)
• Insects & Arthropods (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Oncology (AREA)
• Epidemiology (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Peptides Or Proteins (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Priority Applications (11)

Applications Claiming Priority (2)

Publications (1)

Family

ID=38123823

Family Applications (1)

Country Status (16)

Cited By (3)

Families Citing this family (3)

Citations (3)

• 2006
  • 2006-02-24 CU CU20060047A patent/CU23582A1/es unknown
• 2007
  • 2007-02-22 AR ARP070100749A patent/AR059608A1/es not_active Application Discontinuation
  • 2007-02-23 AT AT07711100T patent/ATE493434T1/de not_active IP Right Cessation
  • 2007-02-23 EP EP07711100A patent/EP1992638B1/en active Active
  • 2007-02-23 MX MX2008010893A patent/MX2008010893A/es active IP Right Grant
  • 2007-02-23 CA CA2638828A patent/CA2638828C/en not_active Expired - Fee Related
  • 2007-02-23 KR KR1020087023166A patent/KR101456205B1/ko not_active Expired - Fee Related
  • 2007-02-23 US US12/278,990 patent/US8283324B2/en not_active Expired - Fee Related
  • 2007-02-23 CN CN2007800138842A patent/CN101426813B/zh not_active Expired - Fee Related
  • 2007-02-23 AU AU2007218671A patent/AU2007218671B2/en not_active Ceased
  • 2007-02-23 ES ES07711100T patent/ES2358845T3/es active Active
  • 2007-02-23 BR BRPI0708148A patent/BRPI0708148B1/pt not_active IP Right Cessation
  • 2007-02-23 WO PCT/CU2007/000006 patent/WO2007095867A1/es not_active Ceased
  • 2007-02-23 JP JP2008555601A patent/JP5069250B2/ja not_active Expired - Fee Related
  • 2007-02-23 RU RU2008137968/04A patent/RU2430109C2/ru not_active IP Right Cessation
• 2008
  • 2008-08-14 ZA ZA200807034A patent/ZA200807034B/xx unknown

Patent Citations (5)

Non-Patent Citations (17)

Also Published As

Similar Documents

Legal Events